Microalgae fermentation using controlled illumination

ABSTRACT

Bioreactors and methods for cultivating microalgae are provided herein. The bioreactor and methods include features and modifications to improve heterotrophic growth efficiency by providing a light signal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/243,593, filed Sep. 18, 2009, and U.S. Provisional Application No.61/359,726, filed Jun. 29, 2010, the entire disclosures of which arehereby incorporated by reference in their entirety for all purposes.

FIELD

The invention relates to methods, means, and systems of fermentation ofmicroorganisms, e.g., microalgae. The invention can be used inpharmaceutical, cosmetic and food industries, as well as for obtainingoil and biofuel from microalgae.

BACKGROUND

Recently, attention has been directed to the application of microalgaeto the production of a variety of materials including lipids,hydrocarbons, oil, polysaccharides, pigments, and biofuels.

One of the conventional methods to grow microalgae is toheterotrophically culture it in an enclosed, light-free system.Techniques have been developed for the large-scale production of aquaticmicroalgae under heterotrophic growth conditions by utilizing organiccarbon instead of light as an energy source. For example, U.S. Pat. Nos.3,142,135 and 3,882,635 describe processes for the heterotrophicproduction of proteins and pigments from algae such as Chlorella,Spongiococcum, and Prototheca. In addition, heterotrophic algal culturescan attain much higher densities than photoautotrophic cultures.

However, the above application cannot be applied to all microalgaebecause only a limited number of microalgae strains can grow inheterotrophic conditions. Attempts to grow microalgae in heterotrophicconditions often involve either screening for strains that can grow inheterotrophic conditions or genetic modification of organisms to allowgrowth under such conditions.

Microalgae that contain both a proper transportation system for sugarand that can grow naturally in heterotrophic conditions often show slowgrowth rates or low production of materials of commercial interest sincethey have evolved many years to utilize sunlight as an environmentalsignal to control aspects of metabolism as well as energy generatedthrough photosynthesis.

Most of the photosynthetic organisms, including microalgae, use light asan environmental signal to optimize themselves for growth and survival.Light signals are sensed by different photoreceptors includingred/far-red photoreceptors (phytochromes) and blue light photoreceptors(cryptochromes and NPHs). Light serves as an environmental signal thatregulates physiological and developmental processes and provides theenergy that fuels the reduction of inorganic carbon. However, undercertain conditions light also has the potential to be toxic.Photoinhibition occurs either when the photon flux absorbed bychloroplasts is very high (under high light conditions) or when thelight energy influx exceeds the consumption capability (undermixotrophic conditions where a cell uses reduced carbon as an energysource). In mixotrophic conditions, photosynthetic organisms showphotoinhibition at a much lower light intensity than autotrophicconditions since the electrons absorbed through the photosyntheticapparatus cannot be efficiently used due to a feedback mechanism in theCalvin cycle.

Absorbed light energy can result in the accumulation of excitedchlorophyll molecules within the pigment bed and damage of thephotosystem. Excited chlorophyll molecules that accumulate in thepigment bed as a consequence of excess excitation can also stimulate theproduction of active oxygen species such as superoxides, hydroxylradicals and singlet oxygen.

SUMMARY

Disclosed herein is a method for cultivating a microalgae capable ofheterotrophic growth, including: incubating the microalgae under aheterotrophic growth condition for a period of time sufficient to allowthe microalgae to grow, wherein the heterotrophic growth conditionincludes a media including a carbon source, and wherein theheterotrophic growth condition further includes a low irradiance oflight.

In some embodiments, the microalgae is a Botryococcus strain, the carbonsource is glucose, and the low irradiance of light is between 1-10 μmolphotons m⁻²s⁻¹.

In some embodiments, the microalgae is a Botryococcus sudeticus strain.In some embodiments, the microalgae is a Botryococcus strain. In someembodiments, the microalgae is a UTEX 2629 strain. In some embodiments,the microalgae is a Botryococcus braunii strain. In some embodiments,the microalgae is a UTEX 2441 strain. In some embodiments, themicroalgae is a Neochloris oleabundans strain. In some embodiments, themicroalgae is a Neochloris strain. In some embodiments, the microalgaeis a UTEX 1185 strain. In some embodiments, the microalgae is aChlamydomonas reinhardtii strain. In some embodiments, the microalgae isa Chlamydomonas strain. In some embodiments, the microalgae is a UTEX2243 strain. In some embodiments, the microalgae comprises aphotoreceptor.

In some embodiments, the carbon source is glucose. In some embodiments,the carbon source is selected from the group consisting of a fixedcarbon source, glucose, fructose, sucrose, galactose, xylose, mannose,rhamnose, N-acetylglucosamine, glycerol, floridoside, glucuronic acid,corn starch, depolymerized cellulosic material, sugar cane, sugar beet,lactose, milk whey, and molasses.

In some embodiments, the light is produced by a natural light source. Insome embodiments, the light is natural sun light. In some embodiments,the light comprises full spectrum light or a specific wavelength oflight. In some embodiments, the light is produced by an artificial lightsource. In some embodiments, the light is artificial light. In someembodiments, the intensity of the low irradiance of light is between0.01-1 μmol photons m⁻² s⁻¹. In some embodiments, the intensity of thelow irradiance of light is between 1-10 μmol photons m⁻²s⁻¹. In someembodiments, the intensity of the low irradiance of light is between10-100 μmol photons m⁻²s⁻¹. In some embodiments, the intensity of thelow irradiance of light is between 100-300 μmol photons m²s⁻¹. In someembodiments, the intensity of the low irradiance of light is between100-300 μmol photons m⁻²s⁻¹. In some embodiments, the intensity of thelow irradiance of light is 3-4 μmol/m²s⁻¹ photons, 2-3 μmol/m²s⁻¹photons, 1-2 μmol/m²s⁻¹ photons, or 3-5 μmol/m²s⁻¹ photons.

In some embodiments, the method further includes producing a materialfrom the microalgae. In some embodiments, the material is apolysaccharide, a pigment, a lipid, or a hydrocarbon. In someembodiments, the material is a hydrocarbon.

In some embodiments, the method further includes recovering thematerial. In some embodiments, the method further includes extractingthe material.

In some embodiments, the method further includes processing thematerial. In some embodiments, the processing of the material produces aprocessed material. In some embodiments, the processed material isselected from the group consisting of a fuel, biodiesel, jet fuel, acosmetic, a pharmaceutical agent, a surfactant, and a renewable diesel.

In some embodiments, the growth rate of the microalgae in the abovemethods is higher than a second microalgae incubated under a secondheterotrophic growth condition for a period of time sufficient to allowthe microalgae to grow, wherein the second heterotrophic growthcondition includes a growth media comprising a carbon source, andwherein the second heterotrophic growth condition does not include a lowirradiance of light.

Also described herein is a method of culturing microalgae, includingplacing a plurality of microalgae cells in the presence of a carbonsource and a low irradiance of light.

Also described herein is a method of manufacturing a material,including: providing a microalgae capable of producing the material;culturing the microalgae in a media, wherein the media includes a carbonsource; applying a low irradiance of light to the microalgae; andallowing the microalgae to accumulate at least 10% of its dry cellweight as the material. In some embodiments, the method further includesrecovering the material.

Also described herein is a bioreactor system, including: a bioreactor; aculture media including a carbon source, wherein the culture media islocated inside the bioreactor; a microalgae adapted for heterotrophicgrowth, wherein the microalgae is located in the culture media; and alight source, wherein the light source produces a low irradiance oflight, and wherein the light source is operatively coupled to thebioreactor.

In some embodiments, light from the light source includes full spectrumlight or a specific wavelength of light. In some embodiments, light fromthe light source includes natural sunlight collected by a solar energycollector operatively coupled to the bioreactor, and wherein the lightis transmitted to the interior of the bioreactor through an opticalfiber operatively coupled to the solar collector and the bioreactor. Insome embodiments, light from the light source includes artificial light,wherein the artificial light is produced by a light emitted diode (LED)or a fluorescent light. In some embodiments, the system further includesa power supply sufficient to power the LED or fluorescent light, whereinthe power supply is operatively coupled to the bioreactor; and a lightcontroller operatively coupled to the power supply, wherein the lightcontroller is adapted to control the intensity and wavelength of lightemitted by the LED or fluorescent light. In some embodiments, theoptical fibre is mounted in a transparent and protective lightstructure. In some embodiments, the LED is mounted in a transparent andprotective light structure.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 is one embodiment of a bioreactor.

FIG. 2 illustrates the isoprenoid/carotenoid pathway.

FIGS. 3A-C show the growth of UTEX 1185 under white, blue, and red lightconditions. The X-axis is shown in days. Glu stands for glucose.

FIGS. 4A-C show the growth of UTEX 2629 under white, blue, and red lightconditions. The X-axis is shown in days. Glu stands for glucose.

FIGS. 5A-C show the growth of UTEX 2441 under white, blue, and red lightconditions. Glu stands for glucose.

FIG. 6 shows the production of lipid by UTEX 2441 under red lightconditions. LG stands for light+glucose; DG stands for dark+glucose.

FIG. 7 shows the growth of UTEX 2243 under white light conditions.

DETAILED DESCRIPTION

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

“Axenic” means a culture of an organism that is free from contaminationby other living organisms.

“Biodiesel” is a biologically produced fatty acid alkyl ester suitablefor use as a fuel in a diesel engine.

The term “biomass” refers to material produced by growth and/orpropagation of cells. Biomass may contain cells and/or intracellularcontents as well as extracellular material. Extracellular materialincludes, but is not limited to, compounds secreted by a cell.

“Bioreactor” means an enclosure or partial enclosure in which cells arecultured, optionally in suspension. FIG. 1 is an example of abioreactor. “Photobioreactor” refers to a container, at least part ofwhich is at least partially transparent or partially open, therebyallowing light to pass through, in which one or more microalgae cellsare cultured. Photobioreactors may be closed, as in the instance of apolyethylene bag or Erlenmeyer flask, or may be open to the environment,as in the instance of an outdoor pond.

As used herein, a “catalyst” refers to an agent, such as a molecule ormacromolecular complex, capable of facilitating or promoting a chemicalreaction of a reactant to a product without becoming a part of theproduct. A catalyst thus increases the rate of a reaction, after which,the catalyst may act on another reactant to form the product. A catalystgenerally lowers the overall activation energy required for the reactionsuch that it proceeds more quickly or at a lower temperature. Thusreaction equilibrium may be more quickly attained. Examples of catalystsinclude enzymes, which are biological catalysts, heat, which is anon-biological catalyst, and metal catalysts used in fossil oil refiningprocesses.

“Cellulosic material” means the products of digestion of cellulose,including glucose and xylose, and optionally additional compounds suchas disaccharides, oligosaccharides, lignin, furfurals and othercompounds. Nonlimiting examples of sources of cellulosic materialinclude sugar cane bagasses, sugar beet pulp, corn stover, wood chips,sawdust and switchgrass.

The term “co-culture”, and variants thereof such as “co-cultivate”,refer to the presence of two or more types of cells in the samebioreactor. The two or more types of cells may both be microorganisms,such as microalgae, or may be a microalgal cell cultured with adifferent cell type. The culture conditions may be those that fostergrowth and/or propagation of the two or more cell types or those thatfacilitate growth and/or proliferation of one, or a subset, of the twoor more cells while maintaining cellular growth for the remainder.

The term “cofactor” is used herein to refer to any molecule, other thanthe substrate, that is required for an enzyme to carry out its enzymaticactivity.

The term “cultivated”, and variants thereof, refer to the intentionalfostering of growth (increases in cell size, cellular contents, and/orcellular activity) and/or propagation (increases in cell numbers viamitosis) of one or more cells by use of intended culture conditions. Thecombination of both growth and propagation may be termed proliferation.The one or more cells may be those of a microorganism, such asmicroalgae. Examples of intended conditions include the use of a definedmedium (with known characteristics such as pH, ionic strength, andcarbon source), specified temperature, oxygen tension, carbon dioxidelevels, and growth in a bioreactor. The term does not refer to thegrowth or propagation of microorganisms in nature or otherwise withoutdirect human intervention, such as natural growth of an organism thatultimately becomes fossilized to produce geological crude oil.

As used herein, the term “cytolysis” refers to the lysis of cells in ahypotonic environment. Cytolysis is caused by excessive osmosis, ormovement of water, towards the inside of a cell (hyperhydration). Thecell cannot withstand the osmotic pressure of the water inside, and soit explodes.

As used herein, the terms “expression vector” or “expression construct”refer to a nucleic acid construct, generated recombinantly orsynthetically, with a series of specified nucleic acid elements thatpermit transcription of a particular nucleic acid in a host cell. Theexpression vector can be part of a plasmid, virus, or nucleic acidfragment. Typically, the expression vector includes a nucleic acid to betranscribed operably linked to a promoter.

“Exogenous gene” refers to a nucleic acid transformed into a cell. Atransformed cell may be referred to as a recombinant cell, into whichadditional exogenous gene(s) may be introduced. The exogenous gene maybe from a different species (and so heterologous), or from the samespecies (and so homologous) relative to the cell being transformed. Inthe case of a homologous gene, it occupies a different location in thegenome of the cell relative to the endogenous copy of the gene. Theexogenous gene may be present in more than one copy in the cell. Theexogenous gene may be maintained in a cell as an insertion into thegenome or as an episomal molecule.

“Exogenously provided” describes a molecule provided to the culturemedia of a cell culture.

“Fixed carbon source” means molecule(s) containing carbon, e.g. organic,that are present at ambient temperature and pressure in solid or liquidform.

“Homogenate” means biomass that has been physically disrupted.

As used herein, “hydrocarbon” refers to: (a) a molecule containing onlyhydrogen and carbon atoms wherein the carbon atoms are covalently linkedto form a linear, branched, cyclic, or partially cyclic backbone towhich the hydrogen atoms are attached; or (b) a molecule that onlyprimarily contains hydrogen and carbon atoms and that can be convertedto contain only hydrogen and carbon atoms by one to four chemicalreactions. Nonlimiting examples of the latter include hydrocarbonscontaining an oxygen atom between one carbon and one hydrogen atom toform an alcohol molecule, as well as aldehydes containing a singleoxygen atom. Methods for the reduction of alcohols to hydrocarbonscontaining only carbon and hydrogen atoms are well known. Anotherexample of a hydrocarbon is an ester, in which an organic group replacesa hydrogen atom (or more than one) in an oxygen acid. The molecularstructure of hydrocarbon compounds varies from the simplest, in the formof methane (CH₄), which is a constituent of natural gas, to the veryheavy and very complex, such as some molecules such as asphaltenes foundin crude oil, petroleum, and bitumens. Hydrocarbons may be in gaseous,liquid, or solid form, or any combination of these forms, and may haveone or more double or triple bonds between adjacent carbon atoms in thebackbone. Accordingly, the term includes linear, branched, cyclic, orpartially cyclic alkanes, alkenes, lipids, and paraffin. Examplesinclude propane, butane, pentane, hexane, octane, triolein, andsqualene.

The term “hydrogen:carbon ratio” refers to the ratio of hydrogen atomsto carbon atoms in a molecule on an atom-to-atom basis. The ratio may beused to refer to the number of carbon and hydrogen atoms in ahydrocarbon molecule. For example, the hydrocarbon with the highestratio is methane CH₄ (4:1).

“Hydrophobic fraction” refers to the portion, or fraction, of a materialthat is more soluble in a hydrophobic phase in comparison to an aqueousphase. A hydrophobic fraction is substantially insoluble in water andusually non-polar.

As used herein, the phrase “increase lipid yield” refers to an increasein the productivity of a microbial culture by, for example, increasingdry weight of cells per liter of culture, increasing the percentage ofcells that constitute lipid, or increasing the overall amount of lipidper liter of culture volume per unit time.

An “inducible promoter” is one that mediates transcription of anoperably linked gene in response to a particular stimulus.

As used herein, the phrase “in operable linkage” refers to a functionallinkage between two sequences, such a control sequence (typically apromoter) and the linked sequence. A promoter is in operable linkagewith an exogenous gene if it can mediate transcription of the gene.

The term “in situ” means “in place” or “in its original position”. Forexample, a culture may contain a first microalga secreting a catalystand a second microorganism secreting a substrate, wherein the first andsecond cell types produce the components necessary for a particularchemical reaction to occur in situ in the co-culture without requiringfurther separation or processing of the materials.

A “limiting concentration of a nutrient” is a concentration in a culturethat limits the propagation of a cultured organism. A “non-limitingconcentration of a nutrient” is a concentration that supports maximalpropagation during a given culture period. Thus, the number of cellsproduced during a given culture period is lower in the presence of alimiting concentration of a nutrient than when the nutrient isnon-limiting. A nutrient is said to be “in excess” in a culture, whenthe nutrient is present at a concentration greater than that whichsupports maximal propagation.

As used herein, a “lipase” is a water-soluble enzyme that catalyzes thehydrolysis of ester bonds in water-insoluble, lipid substrates. Lipasescatalyze the hydrolysis of lipids into glycerols and fatty acids.

“Lipids” are a class of hydrocarbon that are soluble in nonpolarsolvents (such as ether and chloroform) and are relatively or completelyinsoluble in water. Lipid molecules have these properties because theyconsist largely of long hydrocarbon tails which are hydrophobic innature. Examples of lipids include fatty acids (saturated andunsaturated); glycerides or glycerolipids (such as monoglycerides,diglycerides, triglycerides or neutral fats, and phosphoglycerides orglycerophospholipids); nonglycerides (sphingolipids, sterol lipidsincluding cholesterol and steroid hormones, prenol lipids includingterpenoids, fatty alcohols, waxes, and polyketides); and complex lipidderivatives (sugar-linked lipids, or glycolipids, and protein-linkedlipids). “Fats” are a subgroup of lipids called “triacylglycerides.”

The term “low irradiance of light” refers to the irradiance of lightthat can be applied to a microorganism while avoiding significantphotoinhibition under heterotrophic conditions and the irradiance oflight needed to initiate a light-activated metabolism in themicroorganism. Light-activated metabolisms include, but are not limitedto, a life cycle, a circadian rhythm, cell division, a biosyntheticpathway, and a transport system.

As used herein, the term “lysate” refers to a solution containing thecontents of lysed cells.

As used herein, the term “lysis” refers to the breakage of the plasmamembrane and optionally the cell wall of a biological organismsufficient to release at least some intracellular content, often bymechanical, viral or osmotic mechanisms that compromise its integrity.

As used herein, the term “lysing” refers to disrupting the cellularmembrane and optionally the cell wall of a biological organism or cellsufficient to release at least some intracellular content.

“Microalgae” means a eukaryotic microbial organism that contains achloroplast, and optionally that is capable of performingphotosynthesis, or a prokaryotic microbial organism capable ofperforming photosynthesis. Microalgae include obligate photoautotrophs,which cannot metabolize a fixed carbon source as energy, as well asheterotrophs, which can live solely off of a fixed carbon source.Microalgae can refer to unicellular organisms that separate from sistercells shortly after cell division, such as Chlamydomonas, and can alsorefer to microbes such as, for example, Volvox, which is a simplemulticellular photosynthetic microbe of two distinct cell types.“Microalgae” can also refer to cells such as Chlorella and Dunaliella.“Microalgae” also includes other microbial photosynthetic organisms thatexhibit cell-cell adhesion, such as Agmenellum, Anabaena, andPyrobotrys. “Microalgae” also includes obligate heterotrophicmicroorganisms that have lost the ability to perform photosynthesis,such as certain dinoflagellate algae species. Other examples ofmicroalgae are described below.

The terms “microorganism” and “microbe” are used interchangeably hereinto refer to microscopic unicellular organisms, e.g., microalgae.

As used herein, the term “osmotic shock” refers to the rupture of cellsin a solution following a sudden reduction in osmotic pressure. Osmoticshock is sometimes induced to release cellular components of such cellsinto a solution.

“Polysaccharides” (also called “glycans”) are carbohydrates made up ofmonosaccharides joined together by glycosidic linkages. Cellulose is anexample of a polysaccharide that makes up certain plant cell walls.Cellulose can be depolymerized by enzymes to yield monosaccharides suchas xylose and glucose, as well as larger disaccharides andoligosaccharides.

“Port”, in the context of a bioreactor, refers to an opening in thebioreactor that allows influx or efflux of materials such as gases,liquids, and cells. Ports are usually connected to tubing leading fromthe bioreactor.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

As used herein, the term “recombinant” when used with reference, e.g.,to a cell, or nucleic acid, protein, or vector, indicates that the cell,nucleic acid, protein or vector, has been modified by the introductionof an exogenous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as depicted above.

As used herein, the term “renewable diesel” refers to alkanes (such asC:10:0, C12:0, C:14:0, C16:0 and C18:0) produced through hydrogenationand deoxygenation of lipids.

As used herein, the term “sonication” refers to a process of disruptingbiological materials, such as a cell, by use of sound wave energy.

“Species of furfural” refers to 2-Furancarboxaldehyde or a derivativethereof which retains the same basic structural characteristics.

As used herein, “stover” refers to the dried stalks and leaves of a cropremaining after a grain has been harvested.

“Wastewater” is watery waste which typically contains washing water,laundry waste, feces, urine and other liquid or semi-liquid wastes. Itincludes some forms of municipal waste as well as secondarily treatedsewage.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Microorganisms

Any species of organism that produces suitable lipid or hydrocarbon canbe used, although microorganisms that naturally produce high levels ofsuitable lipid or hydrocarbon are preferred. Production of hydrocarbonsby microorganisms is reviewed by Metzger et al. Appl MicrobiolBiotechnol (2005) 66: 486-496 and A Look Back at the U.S. Department ofEnergy's Aquatic Species Program: Biodiesel from Algae,NREL/TP-580-24190, John Sheehan, Terri Dunahay, John Benemann and PaulRoessler (1998).

Considerations affecting the selection of microorganisms for use in theinvention include, in addition to production of suitable lipids orhydrocarbons for production of oils, fuels, and oleochemicals, include:(1) high lipid content as a percentage of cell weight; (2) ease ofgrowth; (3) ease of genetic engineering; and (4) ease of biomassprocessing. In particular embodiments, the wild-type or geneticallyengineered microorganism yields cells that are at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least70% or more lipid. Preferred organisms grow heterotrophically or can beengineered to do so using, for example, methods disclosed herein. Theease of transformation and availability of selectable markers andpromoters, constitutive and/or inducible, that are functional in themicroorganism affect the ease of genetic engineering. Processingconsiderations can include, for example, the availability of effectivemeans for lysing the cells.

In one embodiment, microorganisms include natural or engineeredmicroorganisms that can grow under heterotrophic condition and use lightas signal to control cellular processes. These can include alga such asCyanophyta, Chlorophyta, Rhodophyta, Cryptophyta, Chlorarachniophyta,Haptophyta, Euglenophyta, Heterokontophyta, and Diatoms.

Algae

In one embodiment of the present invention, the microorganism is amicroalgae. Nonlimiting examples of microalgae that can be used inaccordance with the present invention are described below.

More specifically, algal taxa belonging to the Cyanophyta, includingCyanophyceae, are those being Prokaryotae, which have the ability ofoxygen evolution-type photosynthesis and are classified into thefollowing orders and families. Chroococcales include Microcystaceae,Chroococcaceae, Entophysalidaceae, Chamaesiphoniaceae,Dermocarpellaceae, Xenococcaceae, and Hydrococcaceae, Oscillatorialesincludes Borziaceae, Pseudanabaenaceae, Schizotrichaceae, Phormidiaceae,Oscillatoriaceae, and Homoeotrichaceae, Nostocales includesScytonemataceae, Microchaetaceae, Rivulariaceae, and Nostocaceae, andStigonematales includes Chlorogloeopsaceae, Capsosiraceae,Stigonemataceae, Fischerellaceae Borzinemataceae, Nostochopsaceae, andMastigocladaceae.

Chlorophyta include Chlorophyceae, Prasinophyceae, Pedinophyceae,Trebouxiophyceae, and Ulvophyceae. More specifically, Chlorophyceaeincludes Acetabularia, Acicularia, Actinochloris, Amphikrikos,Anadyomene, Ankistrodesmus, Ankyra, Aphanochaete, Ascochloris,Asterococcus, Asteromonas, Astrephomene, Atractomorpha, Axilococcus,Axilosphaera, Basichlamys, Basicladia, Binuclearia, Bipedinomonas,Blastophysa, Boergesenia, Boodlea, Borodinella, Borodinellopsis,Botryococcus, Brachiomonas, Bracteacoccus, Bulbochaete, Caespitella,Capsosiphon, Carteria, Centrosphaera, Chaetomorpha, Chaetonema,Chaetopeltis, Chaetophora, Chalmasia, Chamaetrichon, Characiochloris,Characiosiphon, Characium, Chlamydella, Chlamydobotrys, Chlamydocapsa,Chlamydomonas, Chlamydopodium, Chloranomala, Chlorochydridion,Chlorochytrium, Chlorocladus, Chlorocloster, Chlorococcopsis,Chlorococcum, Chlorogonium, Chloromonas, Chlorophysalis, Chlorosarcina,Chlorosarcinopsis, Chlorosphaera, Chlorosphaeropsis, Chlorotetraedron,Chlorothecium, Chodatella, Choricystis, Cladophora, Cladophoropsis,Cloniophora, Closteriopsis, Coccobotrys, Coelastrella, Coelastropsis,Coelastrum, Coenochloris, Coleochlamys, Coronastrum, Crucigenia,Crucigeniella, Ctenocladus, Cylindrocapsa, Cylindrocapsopsis,Cylindrocystis, Cymopolia, Cystococcus, Cystomonas, Dactylococcus,Dasycladus, Deasonia, Derbesia, Desmatractum, Desmodesmus, Desmotetra,Diacanthos, Dicellula, Dicloster, Dicranochaete, Dictyochloris,Dictyococcus, Dictyosphaeria, Dictyosphaerium, Didymocystis,Didymogenes, Dilabifilum, Dimorphococcus, Diplosphaera, Draparnaldia,Dunaliella, Dysmorphococcus, Echinocoleum, Elakatothrix, Enallax,Entocladia, Entransia, Eremosphaera, Ettlia, Eudorina, Fasciculochloris,Fernandinella, Follicularia, Fottea, Franceia, Friedmannia,Fritschiella, Fusola, Geminella, Gloeococcus, Gloeocystis, Gloeodendron,Gloeomonas, Gloeotila, Golenkinia, Gongrosira, Gonium, Graesiella,Granulocystis, Gyorffiana, Haematococcus, Hazenia, Helicodictyon,Hemichloris, Heterochlamydomonas, Heteromastix, Heterotetracystis,Hormidiospora, Hormidium, Hormotila, Hormotilopsis, Hyalococcus,Hyalodiscus, Hyalogonium, Hyaloraphidium, Hydrodictyon, Hypnomonas,Ignatius, Interfilum, Kentrosphaera, Keratococcus, Kermatia,Kirchneriella, Koliella, Lagerheimia, Lautosphaeria, Leptosiropsis,Lobocystis, Lobomonas, Lola, Macrochloris, Marvania, Micractinium,Microdictyon, Microspora, Monoraphidium, Muriella, Mychonastes,Nanochlorum, Nautococcus, Neglectella, Neochloris, Neodesmus, Neomeris,Neospongiococcum, Nephrochlamys, Nephrocytium, Nephrodiella,Oedocladium, Oedogonium, Oocystella, Oocystis, Oonephris, Ourococcus,Pachycladella, Palmella, Palmellococcus, Palmellopsis, Palmodictyon,Pandorina, Paradoxia, Parietochloris, Pascherina, Paulschulzia,Pectodictyon, Pediastrum, Pedinomonas, Pedinopera, Percursaria,Phacotus, Phaeophila, Physocytium, Pilina, Planctonema, Planktosphaeria,Platydorina, Platymonas, Pleodorina, Pleurastrum, Pleurococcus,Ploeotila, Polyedriopsis, Polyphysa, Polytoma, Polytomella,Prasinocladus, Prasiococcus, Protoderma, Protosiphon,Pseudendocloniopsis, Pseudocharacium, Pseudochlorella,Pseudochlorococcum, Pseudococcomyxa, Pseudodictyosphaerium,Pseudodidymocystis, Pseudokirchneriella, Pseudopleurococcus,Pseudoschizomeris, Pseudoschroederia, Pseudostichococcus,Pseudotetracystis, Pseudotetradron, Pseudotrebouxia, Pteromonas,Pulchrasphaera, Pyramimonas, Pyrobotrys, Quadrigula, Radiofilum,Radiosphaera, Raphidocelis, Raphidonema, Raphidonemopsis, Rhizoclonium,Rhopalosolen, Saprochaete, Scenedesmus, Schizochlamys, Schizomeris,Schroederia, Schroederiella, Scotiellopsis, Siderocystopsis,Siphonocladus, Sirogonium, Sorastrum, Spermatozopsis, Sphaerella,Sphaerellocystis, Sphaerellopsis, Sphaerocystis, Sphaeroplea,Spirotaenia, Spongiochloris, Spongiococcum, Stephanoptera,Stephanosphaera, Stigeoclonium, Struvea, Tetmemorus, Tetrabaena,Tetracystis, Tetradesmus, Tetraedron, Tetrallantos, Tetraselmis,Tetraspora, Tetrastrum, Treubaria, Triploceros, Trochiscia,Trochisciopsis, Ulva, Uronema, Valonia, Valoniopsis, Ventricaria,Viridiella, Vitreochlamys, Volvox, Volvulina, Westella, Willea,Wislouchiella, Zoochlorella, Zygnemopsis, Hyalotheca, Chlorella,Pseudopleurococcum and Rhopalocystis. Prasinophyceae includesHeteromastix, Mammella, Mantoniella, Micromonas, Nephroselmis,Ostreococcus, Prasinocladus, Prasinococcus, Pseudoscourfielda,Pycnococcus, Pyramimonas, Scherffelia. Pedinophyceae includesMarsupiomonas, Pedinomonas, Resultor. Trebouxiophyceae includesApatococcus, Asterochloris, Auxenochlorella, Chlorella, Coccomyxa,Desmococcus, Dictyochloropsis, Elliptochloris, Jaagiella, Leptosira,Lobococcus, Makinoella, Microthamnion, Myrmecia, Nannochloris, Oocystis,Prasiola, Prasiolopsis, Prototheca, Stichococcus, Tetrachlorella,Trebouxia, Trichophilus, Watanabea and Myrmecia. Ulvophyceae includesAcrochaete, Bryopsis, Cephaleuros, Chlorocystis, Enteromorpha,Gloeotilopsis, Halochlorococcum, Ostreobium, Pirula, Pithophora,Planophila, Pseudendoclonium, Trentepohlia, Trichosarcina, Ulothrix,Bolbocoleon, Chaetosiphon, Eugomontia, Oltmannsiellopsis,Pringsheimiella, Pseudodendroclonium, Pseudulvella, Sporocladopsis,Urospora, and Wittrockiella.

Rhodophyta include Acrochaetium, Agardhiella, Antithamnion,Antithamnionella, Asterocytis, Audouinella, Balbiania, Bangia,Batrachospermum, Bonnemaisonia, Bostrychia, Callithamnion, Caloglossa,Ceramium, Champia, Chroodactylon, Chroothece, Compsopogon,Compsopogonopsis, Cumagloia, Cyanidium, Cystoclonium, Dasya, Digenia,Dixoniella, Erythrocladia, Erythrolobas, Erythrotrichia, Flintiella,Galdieria, Gelidium, Glaucosphaera, Goniotrichum, Gracilaria,Grateloupia, Griffithsia, Hildenbrandia, Hymenocladiopsis, Hypnea,Laingia, Membranoptera, Myriogramme, Nemalion, Nemnalionopsis,Neoagardhiella, Palmaria, Phyllophora, Polyneura, Polysiphonia,Porphyra, Porphyridium, Pseudochantransia, Pterocladia, Pugetia,Rhodella, Rhodochaete, Rhodochorton, Rhodosorus, Rhodospora, Rhodymenia,Seirospora, Selenastrum, Sirodotia, Solieria, Spermothamnion, Spyridia,Stylonema, Thorea, Trailiella and Tuomeya.

Cryptophyta include Cryptophycease. More specifically, Campylomonas,Chilomonas, Chroomonas, Cryptochrysis, Cryptomonas, Goniomonas,Guillardia, Hanusia, Hemiselmis, Plagioselmis, Proteomonas, Pyrenomonas,Rhodomonas and Stroreatula.

Chlorarachniophyta include Chlorarachnion, Lotharella and Chattonella.

Haptophyta include Apistonema, Chrysochromulina, Coccolithophora,Corcontochrysis, Cricosphaera, Diacronema, Emiliana, Pavlova, Ruttnera,Cruciplacolithus, Prymnesium, Isochrysis, Calyptrosphaera, Chrysotila,Coccolithus, Dicrateria, Heterosigma, Hymenomonas, Imantonia,Gephyrocapsa, Ochrosphaera, Phaeocystis, Platychrysis, Pseudoisochrysis,Syracosphaera and Pleurochrysis.

Euglenophyta include stasia, Colacium, Cyclidiopsis, Distigma, Euglena,Eutreptia, Eutreptiella, Gyropaigne, Hyalophacus, Khawkinea Astasia,Lepocinclis, Menoidium, Pamidium, Phacus, Rhabdomonas, Rhabdospira,Tetruetreptia and Trachelomonas

Heterokontophyta include Bacillariophyceae, Phaeophyceae, Pelagophyceae,Xanthophyceae, Eustigmatophyceae, Syanurophyceae, Phaeothamniophyceaeand Raphidophyceae. More specifically, Bacillariophyceae includesAchnanthes, Amphora, Chaetoceros, Bacillaria, Nitzschia, Navicula, andPinnularia. Phaeophyceae includes Ascoseira, Asterocladon, Bodanella,Desmarestia, Dictyocha, Dictyota, Ectocarpus, Halopteris, Heribaudiella,Pleurocladia, Porterinema, Pylaiella, Sorocarpus, Spermatochnus,Sphacelaria and Waerniella. Pelagophyceae includes Aureococcus,Aureoumbra, Pelagococcus, Pelagomonas, Pulvinaria and Sarcinochrysis.Xanthophyceae includes Chloramoebales, Rhizochloridales, Mischococcales,Tribonematales, and Vaucheriales. Eustigmatophyceae includesChloridella, Ellipsoidion, Eustigmatos, Monodopsis, Monodus,Nannochloropsis, Polyedriella, Pseudocharaciopsis, Pseudostaurastrum andVischeria Syanurophyceae includes allomonas, Synura and Tessellaria.Phaeothamniophyceae includes haeobotrys and Phaeothamnion.Raphidophyceae includes Olisthodiscus, Vacuolaria and Fibrocapsa.

Diatoms include Bolidophyceae, Coscinodiscophyceae, Dinophyceae andAlveolates. Bolidophyceae include Bolidomonas, Chrysophyceae,Giraudyopsis, Glossomastix, Chromophyton, Chrysamoeba, Chrysochaete,Chrysodidymus, Chrysolepidomonas, Chrysosaccus, Chrysosphaera,Chrysoxys, Cyclonexis, Dinobryon, Epichrysis, Epipyxis, Hibberdia,Lagynion, Lepochromulina, Monas, Monochrysis, Paraphysomonas,Phaeoplaca, Phaeoschizochlamys, Picophagus, Pleurochrysis, Stichogloeaand Uroglena. Coscinodiscophyceae include Bacteriastrum, Bellerochea,Biddulphia, Brockmanniella, Corethron, Coscinodiscus, Eucampia,Extubocellulus, Guinardia, Helicotheca, Leptocylindrus, Leyanella,Lithodesmium, Melosira, Minidiscus, Odontella, Planktoniella, Porosira,Proboscia, Rhizosolenia, Stellarima, Thalassionema, Bicosoecid,Symbiomonas, Actinocyclus, Amphora, Arcocellulus, Detonula, Diatoma,Ditylum, Fragilariophyceae, Asterionellopsis, Delphineis, Grammatophora,Nanofrustulum, Synedra and Tabularia. Dinophyceae includes Adenoides,Alexandrium, Amphidinium, Ceratium, Ceratocorys, Coolia,Crypthecodinium, Exuviaella, Gambierdiscus, Gonyaulax, Gymnodinium,Gyrodinium, Heterocapsa, Katodinium, Lingulodinium, Pfiesteria,Polarella, Protoceratium, Pyrocystis, Scrippsiella, Symbiodinium,Thecadinium, Thoracosphaera, and Zooxanthella. Alveolates includeCystodinium, Glenodinium, Oxyrrhis, Peridinium, Prorocentrum, andWoloszynskia.

Methods of Culturing Microorganisms and Bioreactors

Microorganisms are generally cultured both for purposes of conductinggenetic manipulations and for subsequent production of hydrocarbons(e.g., lipids, fatty acids, aldehydes, alcohols, and alkanes). Theformer type of culture is generally conducted on a small scale andinitially, at least, under conditions in which the startingmicroorganism can grow. Culture for purposes of hydrocarbon productionis usually conducted on a large scale. Preferably a fixed carbon source(e.g. a feedstock) is present. The culture can also be exposed to lightsome or all of the time.

Bioreactor

Microalgae can be cultured in liquid media. The culture can be containedwithin a bioreactor. Microalgae can also be cultured in photobioreactorsthat contain a fixed carbon source and allow light to strike the cells.Exposure of microalgae cells to light, even in the presence of a fixedcarbon source that the cells transport and utilize, can accelerategrowth compared to culturing cells in the dark. Culture conditionparameters can be manipulated to optimize total hydrocarbon production,the combination of hydrocarbon species produced, and/or production of ahydrocarbon species.

FIG. 1 is one embodiment of a bioreactor of the invention. In one aspecta bioreactor is a photobioreactor. In one aspect, a bioreactor systemcan be used for cultivating microalgae. The bioreactor system caninclude a container and an irradiation assembly, where the irradiationassembly is operatively coupled to the container.

In one aspect, a bioreactor is a fermentation tank used for industrialfermentation processes.

In some embodiments, a bioreactor includes glass, metal or plastictanks, equipped with, e.g., gauges and settings to control aeration,stir rate, temperature, pH, and other parameters of interest. Generallythe gauges and settings are operatively coupled to the bioreactor.

In one aspect, a bioreactor can be small enough for bench-topapplications (5-10 L or less) or up to 120,000 L or larger in capacityfor large-scale industrial applications.

In some embodiments, the bioreactor system can include a light-diffusingstructure or a plurality of light-diffusing structures. In someembodiments, one or more of the light-diffusing structures from theplurality of light-diffusing structures are located along the interiorsurface of the bioreactor. In some embodiments, the light-diffusingstructure is operatively coupled to the bioreactor.

The bioreactor system can include one or more optical fibers and/or aplurality of light sources and/or a light source. In some embodiments,the one or more optical fibres are mounted in protective and opticallytransparent lighting structures. In some embodiments, the optical fiberis operatively coupled to the bioreactor. In some embodiments, the lightsource is operatively coupled to the bioreactor.

In some embodiments, the bioreactor system can include a lightingstructure operatively coupled to a bioreactor. In certain embodimentsherein a lighting structure can have any shape or form as it directslight signal to the interior of a bioreactor. The bioreactor system mayalso include at least one optical fiber extending from a first end of atleast one of the one or more optical fibers to a portion of a solarenergy collector. In some embodiments, the solar energy collector isoperatively coupled to the bioreactor. The optical fiber can be adaptedto optically couple the solar energy collector to the bioreactor. Theoptical fiber may be optically coupled (directly or indirectly) to thesolar energy collector.

In some embodiments, the bioreactor system includes a plurality of lightsources operatively coupled to the bioreactor. The plurality of lightsources can include multiple LEDs. The plurality of light sourcescomprising multiple LEDs can be operable to supply full spectrum or aspecific wavelength of artificial light to a bioreactor.

In one embodiment, an LED is mounted in protective and opticallytransparent lighting structures. In one embodiment the LED is an arrayof LEDs.

In some embodiments, microalgae can be grown and maintained in closedbioreactors made of different types of transparent or semitransparentmaterial. Such material can include Plexiglass™ enclosures, glassenclosures, bags made from substances such as polyethylene, transparentor semitransparent pipes, and other materials. Microalgae can be grownand maintained in open bioreactors such as raceway ponds, settlingponds, and other non-enclosed containers.

The gas content of a bioreactor to grow microorganisms like microalgaecan be manipulated. Part of the volume of a bioreactor can contain gasrather than liquid. Gas inlets can be used to pump gases into thebioreactor. Any gas can be pumped into a bioreactor, including air,air/O₂ mixtures, noble gases such as argon and others. The rate of entryof gas into a bioreactor can also be manipulated. Increasing gas flowinto a bioreactor increases the turbidity of a culture of microalgae.Placement of ports conveying gases into a bioreactor can also affect theturbidity of a culture at a given gas flow rate. Air/O₂ mixtures can bemodulated to generate optimal amounts of O₂ for maximal growth by aparticular organism. Microalgae grow significantly faster in the lightunder, for example, 3% O₂/97% air than in 100% air. 3% O₂/97% air isapproximately 100-fold more O₂ than found in air. For example, air:O₂mixtures of about 99.75% air:0.25% O₂, about 99.5% air:0.5% O₂, about99.0% air:1.00% O₂, about 98.0% air:2.0% O₂, about 97.0% air:3.0% O₂,about 96.0% air:4.0% O₂, and about 95.00% air:5.0% O₂ can be infusedinto a bioreactor or bioreactor.

Microalgae cultures can also be subjected to mixing using devices suchas spinning blades and impellers, rocking of a culture, stir bars,infusion of pressurized gas, and other instruments.

Bioreactors can have ports allowing entry of gases, solids, semisolidsand liquids into the chamber containing the microalgae. Ports areusually attached to tubing or other means of conveying substances. Gasports, for example, convey gases into the culture. Pumping gases into abioreactor can serve to both feed cells O₂ and other gases and to aeratethe culture and therefore generate turbidity. The amount of turbidity ofa culture varies as the number and position of gas ports is altered. Forexample, gas ports can be placed along the bottom of a cylindricalpolyethylene bag. Microalgae grow faster when O₂ is added to air andbubbled into a bioreactor.

Bioreactors preferably have one or more ports that allow media entry. Itis not necessary that only one substance enter or leave a port. Forexample, a port can be used to flow culture media into the bioreactorand then later can be used for sampling, gas entry, gas exit, or otherpurposes. In some instances a bioreactor is filled with culture media atthe beginning of a culture and no more growth media is infused after theculture is inoculated. In other words, the microalgal biomass iscultured in an aqueous medium for a period of time during which themicroalgae reproduce and increase in number; however quantities ofaqueous culture medium are not flowed through the bioreactor throughoutthe time period. Thus in some embodiments, aqueous culture medium is notflowed through the bioreactor after inoculation.

In other instances culture media can be flowed though the bioreactorthroughout the time period during which the microalgae reproduce andincrease in number. In some embodiments media is infused into thebioreactor after inoculation but before the cells reach a desireddensity. In other words, a turbulent flow regime of gas entry and mediaentry is not maintained for reproduction of microalgae until a desiredincrease in number of said microalgae has been achieved.

Bioreactors preferably have one or more ports that allow gas entry. Gascan serve to both provide nutrients such as O₂ as well as to provideturbulence in the culture media. Turbulence can be achieved by placing agas entry port below the level of the aqueous culture media so that gasentering the bioreactor bubbles to the surface of the culture. One ormore gas exit ports allow gas to escape, thereby preventing pressurebuildup in the bioreactor. Preferably a gas exit port leads to a“one-way” valve that prevents contaminating microorganisms from enteringthe bioreactor. In some instances cells are cultured in a bioreactor fora period of time during which the microalgae reproduce and increase innumber, however a turbulent flow regime with turbulent eddiespredominantly throughout the culture media caused by gas entry is notmaintained for all of the period of time. In other instances a turbulentflow regime with turbulent eddies predominantly throughout the culturemedia caused by gas entry can be maintained for all of the period oftime during which the microalgae reproduce and increase in number. Insome instances a predetermined range of ratios between the scale of thebioreactor and the scale of eddies is not maintained for the period oftime during which the microalgae reproduce and increase in number. Inother instances such a range can be maintained.

Bioreactors preferably have at least one port that can be used forsampling the culture. Preferably a sampling port can be used repeatedlywithout altering compromising the axenic nature of the culture. Asampling port can be configured with a valve or other device that allowsthe flow of sample to be stopped and started. Alternatively a samplingport can allow continuous sampling. Bioreactors preferably have at leastone port that allows inoculation of a culture. Such a port can also beused for other purposes such as media or gas entry.

In one embodiment, a bioreactor with an irradiation system can be usedto produce hydrocarbon from Botryococcus. Botryococcenes are unbranchedisoprenoid triterpenes having the formula C_(n)H_(2n-10). The A raceproduces alkadienes and alkatrienes (derivatives of fatty acids) whereinn is an odd number 23 through 31. The B race produces botryococceneswherein n is in the range 30 through 40. These can be biofuels of choicefor hydrocracking to gasoline-type hydrocarbons.

Media

Microalgal culture media typically contains components such as a fixednitrogen source, trace elements, optionally a buffer for pH maintenance,and phosphate. Other components can include a fixed carbon source suchas acetate or glucose, and salts such as sodium chloride, particularlyfor seawater microalgae. Examples of trace elements include zinc, boron,cobalt, copper, manganese, and molybdenum in, for example, therespective forms of ZnCl₂, H₃BO₃, CoCl₂.6H₂O, CuCl₂.2H₂O, MnCl₂.4H₂O and(NH₄)₆Mo₇O₂4.4H₂O.

For organisms able to grow on a fixed carbon source, the fixed carbonsource can be, for example, glucose, fructose, sucrose, galactose,xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, floridoside,and/or glucuronic acid. The one or more carbon source(s) can be suppliedat a concentration of less than 50 μM, at least about 50 μM, at leastabout 100 μM, at least about 500 μM, at least about 5 mM, at least about50 mM, at least about 500 mM, and more than 500 mM of one or moreexogenously provided fixed carbon source(s). The one or more carbonsource(s) can be supplied at a less than 1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the media. The one or morecarbon source(s) can also be supplied at a percentage of the mediabetween the above noted percentages, e.g., 2.5% or 3.7% of the media.

Some microorganisms naturally grow on or can be engineered to grow on afixed carbon source that is a heterogeneous source of compounds such asmunicipal waste, secondarily treated sewage, wastewater, and othersources of fixed carbon and other nutrients such as sulfates,phosphates, and nitrates. The sewage component serves as a nutrientsource in the production of hydrocarbons, and the culture provides aninexpensive source of hydrocarbons.

Other culture parameters can also be manipulated, such as the pH of theculture media, the identity and concentration of trace elements andother media constituents.

Microorganisms useful in accordance with the methods of the presentinvention are found in various locations and environments throughout theworld. As a consequence of their isolation from other species and theirresulting evolutionary divergence, the particular growth medium foroptimal growth and generation of lipid and/or hydrocarbon constituentscan vary. In some cases, certain strains of microorganisms may be unableto grow on a particular growth medium because of the presence of someinhibitory component or the absence of some essential nutritionalrequirement required by the particular strain of microorganism.

Solid and liquid growth media are generally available from a widevariety of sources, and instructions for the preparation of particularmedia that is suitable for a wide variety of strains of microorganismscan be found, for example, online at a site maintained by the Universityof Texas at Austin for its culture collection of algae (UTEX).

Process conditions can be adjusted to increase the yield of lipidssuitable for a particular use and/or to reduce production cost. Forexample, in certain embodiments, a microbe (e.g., microalgae) iscultured in the presence of a limiting concentration of one or morenutrients, such as, for example, carbon and/or nitrogen, phosphorous, orsulfur, while providing an excess of fixed carbon energy such asglucose. Nitrogen limitation tends to increase microbial lipid yieldover microbial lipid yield in a culture in which nitrogen is provided inexcess. In particular embodiments, the increase in lipid yield is atleast about: 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or500%. The microbe can be cultured in the presence of a limiting amountof a nutrient for a portion of the total culture period or for theentire period. In particular embodiments, the nutrient concentration iscycled between a limiting concentration and a non-limiting concentrationat least twice during the total culture period.

Heterotrophic Growth and Light

Microorganisms can be cultured under heterotrophic growth conditions inwhich a fixed carbon source provides energy for growth and lipidaccumulation.

Standard methods for the heterotrophic growth and propagation ofmicroalgae are known (see for example Miao and Wu, J Biotechnology,2004, 11:85-93 and Miao and Wu, Biosource Technology (2006) 97:841-846).

For hydrocarbon production, cells, including recombinant cells of theinvention described herein, can be cultured or fermented in largequantities. The culturing may be in large liquid volumes, such as insuspension cultures as an example. Other examples include starting witha small culture of cells which expand into a large biomass incombination with cell growth and propagation as well as hydrocarbonproduction. Bioreactors or steel fermentors can be used to accommodatelarge culture volumes. A bioreactor can include a fermentor. A fermentorsimilar those used in the production of beer and/or wine can besuitable, as are extremely large fermentors used in the production ofethanol.

Appropriate nutrient sources for culture in a fermentor are provided.These include raw materials such as one or more of the following: afixed carbon source such as glucose, corn starch, depolymerizedcellulosic material, sucrose, sugar cane, sugar beet, lactose, milkwhey, or molasses; a fat source, such as fats or vegetable oils; anitrogen source, such as protein, soybean meal, cornsteep liquor,ammonia (pure or in salt form), nitrate or nitrate salt, or molecularnitrogen; and a phosphorus source, such as phosphate salts.Additionally, a fermentor allows for the control of culture conditionssuch as temperature, pH, oxygen tension, and carbon dioxide levels.Optionally, gaseous components, like oxygen or nitrogen, can be bubbledthrough a liquid culture. Other Starch (glucose) sources such as wheat,potato, rice, and sorghum. Other carbon sources include process streamssuch as technical grade glycerol, black liquor, organic acids such asacetate, and molasses. Carbon sources can also be provided as a mixture,such as a mixture of sucrose and depolymerized sugar beet pulp.

A fermentor can be used to allow cells to undergo the various phases oftheir growth cycle. As an example, an inoculum of hydrocarbon-producingcells can be introduced into a medium followed by a lag period (lagphase) before the cells begin growth. Following the lag period, thegrowth rate increases steadily and enters the log, or exponential,phase. The exponential phase is in turn followed by a slowing of growthdue to decreases in nutrients and/or increases in toxic substances.After this slowing, growth stops, and the cells enter a stationary phaseor steady state, depending on the particular environment provided to thecells.

Hydrocarbon production by cells disclosed herein can occur during thelog phase or thereafter, including the stationary phase whereinnutrients are supplied, or still available, to allow the continuation ofhydrocarbon production in the absence of cell division.

Preferably, microorganisms grown using conditions described herein andknown in the art comprise at least about 20% by weight of lipid,preferably at least about 40% by weight, more preferably at least about50% by weight, and most preferably at least about 60% by weight.

In an alternate heterotrophic growth method in accordance with thepresent invention, microorganisms can be cultured using depolymerizedcellulosic biomass as a feedstock. Cellulosic biomass (e.g., stover,such as corn stover) is inexpensive and readily available; however,attempts to use this material as a feedstock for yeast have failed. Inparticular, such feedstock has been found to be inhibitory to yeastgrowth, and yeast cannot use the 5-carbon sugars produced fromcellulosic materials (e.g., xylose from hemi-cellulose). By contrast,microalgae can grow on processed cellulosic material. Accordingly, theinvention provides a method of culturing microalgae in the presence of acellulosic material and/or a 5-carbon sugar.

Suitable cellulosic materials include residues from herbaceous and woodyenergy crops, as well as agricultural crops, i.e., the plant parts,primarily stalks and leaves, not removed from the fields with theprimary food or fiber product. Examples include agricultural wastes suchas sugarcane bagasse, rice hulls, corn fiber (including stalks, leaves,husks, and cobs), wheat straw, rice straw, sugar beet pulp, citrus pulp,citrus peels; forestry wastes such as hardwood and softwood thinnings,and hardwood and softwood residues from timber operations; wood wastessuch as saw mill wastes (wood chips, sawdust) and pulp mill waste; urbanwastes such as paper fractions of municipal solid waste, urban woodwaste and urban green waste such as municipal grass clippings; and woodconstruction waste. Additional cellulosics include dedicated cellulosiccrops such as switchgrass, hybrid poplar wood, and miscanthus, fibercane, and fiber sorghum. Five-carbon sugars that are produced from suchmaterials include xylose.

In still another alternative heterotrophic growth method in accordancewith the present invention, which itself may optionally be used incombination with the methods described above, sucrose, produced byexample from sugar cane or sugar beet, is used as a feedstock.

Heterotrophic growth can include the use of both light and fixed carbonsource(s) for cells to grow and produce hydrocarbons. Heterotrophicgrowth can be conducted in a photobioreactor.

Bioreactors can be exposed to one or more light sources to providemicroalgae with a light signal. A light signal can be provided via lightdirected to a surface of the bioreactor by a light source. Preferablythe light source provides an intensity that is sufficient for the cellsto grow, but not so intense as to cause oxidative damage or cause aphotoinhibitive response. In some instances a light source has awavelength range that mimics or approximately mimics the range of thesun. In other instances a different wavelength range is used.Bioreactors can be placed outdoors or in a greenhouse or other facilitythat allows sunlight to strike the surface. In some embodiments, photonintensities for species of the genus Botryococcus are between 25 and 500μmE m⁻²s⁻¹ (see for example Photosynth Res. 2005 June; 84(1-3):21-7).

The number of photons striking a culture of microalgae cells can bemanipulated, as well as other parameters such as the wavelength spectrumand ratio of dark:light hours per day. Microalgae can also be culturedin natural light, as well as simultaneous and/or alternatingcombinations of natural light and artificial light. For example,microalgae can be cultured under natural light during daylight hours andunder artificial light during night hours.

In one aspect of the invention, a microorganism is exposed to about 0.1%to about 1% of light irradiance required for photosynthesis, preferablyabout 0.3% to about 0.8% of light irradiance required for photosynthesisby the organism. Typical light irradiance can be between 0.1-300 μmolphotons m⁻²s⁻¹ including less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 to99, 100, 101 to 149, 150, 151, to 199, 200, 201 to 249, 250, or greaterthan 250 μmol photons m⁻²s⁻¹. Light irradiance can be about 0.01-1 μmolphotons m²s⁻¹, preferably between 1-10 μmol photons m⁻²s⁻¹, or between10-100 μmol photons m²s⁻¹, or between 100-300 μmol photons m⁻²s⁻¹, orbetween 100-300 μmol photons m²s⁻¹. Also included are light irradiancesbetween the above noted light irradiances, e.g., 1.1, 2.1, 2.5, or 3.5μmol photons m²s⁻¹.

In one aspect, different light spectrums (e.g. 360-700 nm) can be used.Light spectrums can be less than 300, 300, 350, 400, 450, 500, 550, 600,650, 700, or 750 nm or more. Also included are light spectrums betweenthe above noted light spectrums, e.g., 360 or 440 nm.

In one embodiment, the irradiation can be applied in a continuousmanner. In another embodiment, the irradiation can be applied in acyclic pattern with an appropriate period of lighting including but notlimited to 12 h light:12 h dark or 16 h light:8 h dark. Light patternscan include less than 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 h of light and/or less than 1,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, or 23 hours (h) of dark. Also included are light patternsbetween the above noted light patterns, e.g., 7.5 h of light or 7.5 h ofdark.

In one embodiment, the irradiation can be natural sunlight collected bya solar collector and transmitted to the interior of a bioreactorthrough an optical fiber.

In another embodiment, artificial light, such as light emitted diodes(LEDs) or fluorescent light can be used as light source. In anotherembodiment, natural sunlight and artificial light can be used together.

In one embodiment, the irradiation is a full spectrum of light.

In another embodiment, the irradiation is a specific wavelength of lightor a range of spectrum light transmitted through a specific filter.

Methods of Recovering Lipids and Hydrocarbons

Hydrocarbons (e.g., lipids, fatty acids, aldehydes, alcohols, andalkanes) produced by cells of the invention can be harvested, orotherwise collected, by any convenient means. For example, hydrocarbonssecreted from cells can be centrifuged to separate the hydrocarbons in ahydrophobic layer from contaminants in an aqueous layer and optionallyfrom any solid materials as a precipitate in after centrifugation.Material containing cell or cell fractions can be treated with proteasesto degrade contaminating proteins before or after centrifugation. Insome instances the contaminating proteins are associated, possiblycovalently, to hydrocarbons or hydrocarbon precursors which formhydrocarbons upon removal of the protein. In other instances thehydrocarbon molecules are in a preparation that also contains proteins.Proteases can be added to hydrocarbon preparations containing proteinsto degrade proteins (for example, the protease from Streptomyces griseuscan be used (SigmaAldrich catalog number P5147). After digestion, thehydrocarbons are preferably purified from residual proteins, peptidefragments, and amino acids. This purification can be accomplished, forexample, by methods listed above such as centrifugation and filtration.

Extracellular hydrocarbons can also be extracted in vivo from livingmicroalgae cells which are then returned to a bioreactor by exposure ofthe cells, in an otherwise sterile environment, to a non-toxicextraction solvent, followed by separation of the living cells and thehydrophobic fraction of extraction solvent and hydrocarbons, wherein theseparated living cells are then returned to a culture container such asa stainless steel fermentor or photobioreactor (see Biotechnol Bioeng.2004 Dec. 5; 88(5):593-600 and Biotechnol Bioeng. 2004 Mar. 5;85(5):475-81).

Hydrocarbons can also be isolated by whole cell extraction. The cellsare first disrupted and then intracellular and cell membrane/cellwall-associated hydrocarbons as well as extracellular hydrocarbons canbe collected from the whole cell mass, such as by use of centrifugationas described above.

Various methods are available for separating hydrocarbons and lipidsfrom cellular lysates produced by the above methods. For example,hydrocarbons can be extracted with a hydrophobic solvent such as hexane(see Frenz et al. 1989, Enzyme Microb. Technol., 11:717). Hydrocarbonscan also be extracted using liquefaction (see for example Sawayama etal. 1999, Biomass and Bioenergy 17:33-39 and Inoue et al. 1993, BiomassBioenergy 6(4):269-274); oil liquefaction (see for example Minowa et al.1995, Fuel 74(12):1735-1738); and supercritical CO₂ extraction (see forexample Mendes et al. 2003, Inorganica Chimica Acta 356:328-334).

Miao and Wu describe a protocol of the recovery of microalgal lipid froma culture in which the cells were harvested by centrifugation, washedwith distilled water and dried by freeze drying. The resulting cellpowder was pulverized in a mortor and then extracted with n-hexane. Miaoand Wu, Biosource Technology (2006) 97:841-846.

Lysing Cells

Intracellular lipids and hydrocarbons produced in microorganisms are, insome embodiments, extracted after lysing the cells of the microorganism.Once extracted, the lipids and/or hydrocarbons can be further refined toproduce, e.g., oils, fuels, or oleochemicals.

After completion of culturing, the microorganisms can be separated fromthe fermentation broth. Optionally, the separation is effected bycentrifugation to generate a concentrated paste. Centrifugation does notremove significant amounts of intracellular water from themicroorganisms and is not a drying step. The biomass can then be washedwith a washing solution (e.g., DI water) to get rid of the fermentationbroth and debris. Optionally, the washed microbial biomass may also bedried (oven dried, lyophilized, etc.) prior to cell disruption.Alternatively, cells can be lysed without separation from some or all ofthe fermentation broth when the fermentation is complete. For example,the cells can be at a ratio of less than 1:1 v:v cells to extracellularliquid when the cells are lysed.

Microorganisms containing a lipid and/or hydrocarbon can be lysed toproduce a lysate. As detailed herein, the step of lysing a microorganism(also referred to as cell lysis) can be achieved by any convenientmeans, including heat-induced lysis, adding a base, adding an acid,using enzymes such as proteases and polysaccharide degradation enzymessuch as amylases, using ultrasound, mechanical lysis, using osmoticshock, infection with a lytic virus, and/or expression of one or morelytic genes. Lysis is performed to release intracellular molecules whichhave been produced by the microorganism. Each of these methods forlysing a microorganism can be used as a single method or in combinationsimultaneously or sequentially.

The extent of cell disruption can be observed by microscopic analysis.Using one or more of the methods described herein, typically more than70% cell breakage is observed. Preferably, cell breakage is more than80%, more preferably more than 90% and most preferred about 100%.

In particular embodiments, the microorganism is lysed after growth, forexample to increase the exposure of cellular lipid and/or hydrocarbonfor extraction or further processing. The timing of lipase expression(e.g., via an inducible promoter) or cell lysis can be adjusted tooptimize the yield of lipids and/or hydrocarbons. Below are described anumber of lysis techniques. These techniques can be used individually orin combination.

Heat-Induced Lysis

In a preferred embodiment of the present invention, the step of lysing amicroorganism comprises heating of a cellular suspension containing themicroorganism. In this embodiment, the fermentation broth containing themicroorganisms (or a suspension of microorganisms isolated from thefermentation broth) is heated until the microorganisms, i.e., the cellwalls and membranes of microorganisms degrade or breakdown. Typically,temperatures applied are at least 50 C. Higher temperatures, such as, atleast 30 C, at least 60 C, at least 70 C, at least 80 C, at least 90 C,at least 100 C, at least 110 C, at least 120 C, at least 130 C or higherare used for more efficient cell lysis.

Lysing cells by heat treatment can be performed by boiling themicroorganism. Alternatively, heat treatment (without boiling) can beperformed in an autoclave. The heat treated lysate may be cooled forfurther treatment.

Cell disruption can also be performed by steam treatment, i.e., throughaddition of pressurized steam. Steam treatment of microalgae for celldisruption is described, for example, in U.S. Pat. No. 6,750,048.

Lysis Using a Base

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises adding a base to a cellular suspensioncontaining the microorganism.

The base should be strong enough to hydrolyze at least a portion of theproteinaceous compounds of the microorganisms used. Bases which areuseful for solubilizing proteins are known in the art of chemistry.Exemplary bases which are useful in the methods of the present inventioninclude, but are not limited to, hydroxides, carbonates and bicarbonatesof lithium, sodium, potassium, calcium, and mixtures thereof. Apreferred base is KOH. Base treatment of microalgae for cell disruptionis described, for example, in U.S. Pat. No. 6,750,048.

Acidic Lysis

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises adding an acid to a cellular suspensioncontaining the microorganism. Acid lysis can be affected using an acidat a concentration of 10-500 mN or preferably 40-160 nM. Acid lysis ispreferably performed at above room temperature (e.g., at 40-160, andpreferably a temperature of 50-130. For moderate temperatures (e.g.,room temperature to 100 C and particularly room temperature to 65, acidtreatment can usefully be combined with sonication or other celldisruption methods.

Lysing Cells Using Enzymes

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises lysing the microorganism by using anenzyme. Preferred enzymes for lysing a microorganism are proteases andpolysaccharide-degrading enzymes such as hemicellulase (e.g.,hemicellulase from Aspergillus niger; Sigma Aldrich, St. Louis, Mo.;#H2125), pectinase (e.g., pectinase from Rhizopus sp.; Sigma Aldrich,St. Louis, Mo.; #P2401), Mannaway 4.0 L (Novozymes), cellulase (e.g.,cellulose from Trichoderma viride; Sigma Aldrich, St. Louis, Mo.;#C9422), and driselase (e.g., driselase from Basidiomycetes sp.; SigmaAldrich, St. Louis, Mo.; #D9515.

Cellulases

In a preferred embodiment of the present invention, a cellulase forlysing a microorganism is a polysaccharide-degrading enzyme, optionallyfrom Chlorella or a Chlorella virus.

Proteases

Proteases such as Streptomyces griseus protease, chymotrypsin,proteinase K, proteases listed in Degradation of Polylactide byCommercial Proteases, Oda Y et al., Journal of Polymers and theEnvironment, Volume 8, Number 1, January 2000, pp. 29-32(4), and otherproteases can be used to lyse microorganisms. Other proteases that canbe used include Alcalase 2.4 FG (Novozymes) and Flavourzyme 100 L(Novozymes).

Combinations

Any combination of a protease and a polysaccharide-degrading enzyme canalso be used, including any combination of the preceding proteases andpolysaccharide-degrading enzymes.

Lysing Cells Using Ultrasound

In another preferred embodiment of the present invention, the step oflysing a microorganism is performed by using ultrasound, i.e.,sonication. Thus, cells can also by lysed with high frequency sound. Thesound can be produced electronically and transported through a metallictip to an appropriately concentrated cellular suspension. Thissonication (or ultrasonication) disrupts cellular integrity based on thecreation of cavities in cell suspension.

Mechanical Lysis

In another preferred embodiment of the present invention, the step oflysing a microorganism is performed by mechanical lysis. Cells can belysed mechanically and optionally homogenized to facilitate hydrocarbon(e.g., lipid) collection. For example, a pressure disrupter can be usedto pump a cell containing slurry through a restricted orifice valve.High pressure (up to 1500 bar) is applied, followed by an instantexpansion through an exiting nozzle. Cell disruption is accomplished bythree different mechanisms: impingement on the valve, high liquid shearin the orifice, and sudden pressure drop upon discharge, causing anexplosion of the cell. The method releases intracellular molecules.

Alternatively, a ball mill can be used. In a ball mill, cells areagitated in suspension with small abrasive particles, such as beads.Cells break because of shear forces, grinding between beads, andcollisions with beads. The beads disrupt the cells to release cellularcontents. Cells can also be disrupted by shear forces, such as with theuse of blending (such as with a high speed or Waring blender asexamples), the french press, or even centrifugation in case of weak cellwalls, to disrupt cells.

Lysing Cells by Osmotic Shock (Cytolysis)

In another preferred embodiment of the present invention, the step oflysing a microorganism is performed by applying an osmotic shock.

Infection with a Lytic Virus

In a preferred embodiment of the present invention, the step of lysing amicroorganism comprises infection of the microorganism with a lyticvirus. A wide variety of viruses are known to lyse microorganismssuitable for use in the present invention, and the selection and use ofa particular lytic virus for a particular microorganism is within thelevel of skill in the art.

For example, paramecium bursaria chlorella virus (PBCV-1) is theprototype of a group (family Phycodnaviridae, genus Chlorovirus) oflarge, icosahedral, plaque-forming, double-stranded DNA viruses thatreplicate in, and lyse, certain unicellular, eukaryotic chlorella-likegreen algae. Accordingly, any susceptible microalgae can be lysed byinfecting the culture with a suitable chlorella virus. See for exampleAdv. Virus Res. 2006; 66:293-336; Virology, 1999 Apr. 25; 257(1):15-23;Virology, 2004 Jan. 5; 318(1):214-23; Nucleic Acids Symp. Ser. 2000;(44):161-2; J. Virol. 2006 March; 80(5):2437-44; and Annu Rev.Microbiol. 1999; 53:447-94.

Autolysis (Expression of a Lytic Gene)

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises autolysis. In this embodiment, amicroorganism according to the invention is genetically engineered toproduce a lytic protein that will lyse the microorganism. This lyticgene can be expressed using an inducible promoter so that the cells canfirst be grown to a desirable density in a fermentor, followed byinduction of the promoter to express the lytic gene to lyse the cells.In one embodiment, the lytic gene encodes a polysaccharide-degradingenzyme.

In certain other embodiments, the lytic gene is a gene from a lyticvirus. Thus, for example, a lytic gene from a Chlorella virus can beexpressed in an algal cell of the genus Chlorella, such as C.protothecoides.

Suitable expression methods are described herein with respect to theexpression of a lipase gene. Expression of lytic genes is preferablydone using an inducible promoter, such as a promoter active inmicroalgae that is induced by a stimulus such as the presence of a smallmolecule, light, heat, and other stimuli. For example, see Virology 260,308-315 (1999); FEMS Microbiology Letters 180 (1999) 45-53; Virology263, 376-387 (1999); and Virology 230, 361-368 (1997).

Extraction of Lipids and Hydrocarbons

Lipids and hydrocarbons generated by the microorganisms of the presentinvention can be recovered by extraction with an organic solvent. Insome cases, the preferred organic solvent is hexane. Typically, theorganic solvent is added directly to the lysate without prior separationof the lysate components. In one embodiment, the lysate generated by oneor more of the methods described above is contacted with an organicsolvent for a period of time sufficient to allow the lipid and/orhydrocarbon components to form a solution with the organic solvent. Insome cases, the solution can then be further refined to recover specificdesired lipid or hydrocarbon components. Hexane extraction methods arewell known in the art.

Methods of Processing Lipids and Hydrocarbons

Enzymatic Modification

Hydrocarbons (e.g., lipids, fatty acids, aldehydes, alcohols, andalkanes) produced by cells as described herein can be modified by theuse of one or more enzymes, including a lipase. When the hydrocarbonsare in the extracellular environment of the cells, the one or moreenzymes can be added to that environment under conditions in which theenzyme modifies the hydrocarbon or completes its synthesis from ahydrocarbon precursor. Alternatively, the hydrocarbons can be partially,or completely, isolated from the cellular material before addition ofone or more catalysts such as enzymes. Such catalysts are exogenouslyadded, and their activity occurs outside the cell or in vitro.

Thermal and Other Catalytic Modification

Hydrocarbons produced by cells in vivo, or enzymatically modified invitro, as described herein can be optionally further processed byconventional means. The processing can include “cracking” to reduce thesize, and thus increase the hydrogen:carbon ratio, of hydrocarbonmolecules. Catalytic and thermal cracking methods are routinely used inhydrocarbon and triglyceride oil processing. Catalytic methods involvethe use of a catalyst, such as a solid acid catalyst. The catalyst canbe silica-alumina or a zeolite, which result in the heterolytic, orasymmetric, breakage of a carbon-carbon bond to result in a carbocationand a hydride anion. These reactive intermediates then undergo eitherrearrangement or hydride transfer with another hydrocarbon. Thereactions can thus regenerate the intermediates to result in aself-propagating chain mechanism. Hydrocarbons can also be processed toreduce, optionally to zero, the number of carbon-carbon double, ortriple, bonds therein. Hydrocarbons can also be processed to remove oreliminate a ring or cyclic structure therein. Hydrocarbons can also beprocessed to increase the hydrogen:carbon ratio. This can include theaddition of hydrogen (“hydrogenation”) and/or the “cracking” ofhydrocarbons into smaller hydrocarbons.

Thermal methods involve the use of elevated temperature and pressure toreduce hydrocarbon size. An elevated temperature of about 80° C. andpressure of about 700 kPa can be used. These conditions generate“light,” a term that is sometimes used to refer to hydrogen-richhydrocarbon molecules (as distinguished from photon flux), while alsogenerating, by condensation, heavier hydrocarbon molecules which arerelatively depleted of hydrogen. The methodology provides homolytic, orsymmetrical, breakage and produces alkenes, which may be optionallyenzymatically saturated as described above.

Catalytic and thermal methods are standard in plants for hydrocarbonprocessing and oil refining. Thus hydrocarbons produced by cells asdescribed herein can be collected and processed or refined viaconventional means. See Hillen et al. (Biotechnology and Bioengineering,Vol. XXIV: 193-205 (1982)) for a report on hydrocracking ofmicroalgae-produced hydrocarbons. In alternative embodiments, thefraction is treated with another catalyst, such as an organic compound,heat, and/or an inorganic compound. For processing of lipids intobiodiesel, a transesterification process is used as described in SectionIV herein.

Hydrocarbons produced via methods of the present invention are useful ina variety of industrial applications. For example, the production oflinear alkylbenzene sulfonate (LAS), an anionic surfactant used innearly all types of detergents and cleaning preparations, utilizeshydrocarbons generally comprising a chain of 10-14 carbon atoms. See,for example, U.S. Pat. Nos. 6,946,430; 5,506,201; 6,692,730; 6,268,517;6,020,509; 6,140,302; 5,080,848; and 5,567,359. Surfactants, such asLAS, can be used in the manufacture of personal care compositions anddetergents, such as those described in U.S. Pat. Nos. 5,942,479;6,086,903; 5,833,999; 6,468,955; and 6,407,044.

Methods of Producing Fuels Suitable for Use in Diesel Vehicles and JetEngines

Increasing interest is directed to the use of hydrocarbon components ofbiological origin in fuels, such as biodiesel, renewable diesel, and jetfuel, since renewable biological starting materials that may replacestarting materials derived from fossil fuels are available, and the usethereof is desirable. There is a need for methods for producinghydrocarbon components from biological materials. The present inventionfulfills this need by providing methods for production of biodiesel,renewable diesel, and jet fuel using the lipids generated by the methodsdescribed herein as a biological material to produce biodiesel,renewable diesel, and jet fuel.

Traditional diesel fuels are petroleum distillates rich in paraffinichydrocarbons. They have boiling ranges as broad as 370 to 780 F, whichare suitable for combustion in a compression ignition engine, such as adiesel engine vehicle. The American Society of Testing and Materials(ASTM) establishes the grade of diesel according to the boiling range,along with allowable ranges of other fuel properties, such as cetanenumber, cloud point, flash point, viscosity, aniline point, sulfurcontent, water content, ash content, copper strip corrosion, and carbonresidue. Technically, any hydrocarbon distillate material derived frombiomass or otherwise that meets the appropriate ASTM specification canbe defined as diesel fuel (ASTM D975), jet fuel (ASTM D1655), or asbiodiesel (ASTM D6751).

After extraction, lipid and/or hydrocarbon components recovered from themicrobial biomass described herein can be subjected to chemicaltreatment to manufacture a fuel for use in diesel vehicles and jetengines.

Biodiesel

Biodiesel is a liquid which varies in color—between golden and darkbrown—depending on the production feedstock. It is practicallyimmiscible with water, has a high boiling point and low vapor pressure.Biodiesel refers to a diesel-equivalent processed fuel for use indiesel-engine vehicles. Biodiesel is biodegradable and non-toxic. Anadditional benefit of biodiesel over conventional diesel fuel is lowerengine wear.

Typically, biodiesel comprises C14-C18 alkyl esters. Various processesconvert biomass or a lipid produced and isolated as described herein todiesel fuels. A preferred method to produce biodiesel is bytransesterification of a lipid as described herein. A preferred alkylester for use as biodiesel is a methyl ester or ethyl ester.

Biodiesel produced by a method described herein can be used alone orblended with conventional diesel fuel at any concentration in mostmodern diesel-engine vehicles. When blended with conventional dieselfuel (petroleum diesel), biodiesel may be present from about 0.1% toabout 99.9%. Much of the world uses a system known as the “B” factor tostate the amount of biodiesel in any fuel mix. For example, fuelcontaining 20% biodiesel is labeled B20. Pure biodiesel is referred toas B100.

Biodiesel can also be used as a heating fuel in domestic and commercialboilers. Existing oil boilers may contain rubber parts and may requireconversion to run on biodiesel. The conversion process is usuallyrelatively simple, involving the exchange of rubber parts for syntheticparts due to biodiesel being a strong solvent. Due to its strong solventpower, burning biodiesel will increase the efficiency of boilers.

Biodiesel can be used as an additive in formulations of diesel toincrease the lubricity of pure Ultra-Low Sulfur Diesel (ULSD) fuel,which is advantageous because it has virtually no sulfur content.

Biodiesel is a better solvent than petrodiesel and can be used to breakdown deposits of residues in the fuel lines of vehicles that havepreviously been run on petrodiesel.

Production of Biodiesel

Biodiesel can be produced by transesterification of triglyceridescontained in oil-rich biomass. Thus, in another aspect of the presentinvention a method for producing biodiesel is provided. In a preferredembodiment, the method for producing biodiesel comprises the steps of(a) cultivating a lipid-containing microorganism using methods disclosedherein (b) lysing a lipid-containing microorganism to produce a lysate,(c) isolating lipid from the lysed microorganism, and (d)transesterifying the lipid composition, whereby biodiesel is produced.

Methods for growth of a microorganism, lysing a microorganism to producea lysate, treating the lysate in a medium comprising an organic solventto form a heterogeneous mixture and separating the treated lysate into alipid composition have been described above and can also be used in themethod of producing biodiesel.

Lipid compositions can be subjected to transesterification to yieldlong-chain fatty acid esters useful as biodiesel. Preferredtransesterification reactions are outlined below and include basecatalyzed transesterification and transesterification using recombinantlipases.

In a base-catalyzed transesterification process, the triacylglyceridesare reacted with an alcohol, such as methanol or ethanol, in thepresence of an alkaline catalyst, typically potassium hydroxide. Thisreaction forms methyl or ethyl esters and glycerin (glycerol) as abyproduct.

General Chemical Process

Animal and plant oils are typically made of triglycerides which areesters of free fatty acids with the trihydric alcohol, glycerol. Intransesterification, the glycerol in a triacylglyceride (TAG) isreplaced with a short-chain alcohol such as methanol or ethanol.

Using Recombinant Lipases

Transesterification has also been carried out experimentally using anenzyme, such as a lipase instead of a base. Lipase-catalyzedtransesterification can be carried out, for example, at a temperaturebetween the room temperature and 80 C, and a mole ratio of the TAG tothe lower alcohol of greater than 1:1, preferably about 3:1.

Lipases suitable for use in transesterification are found in, e.g. U.S.Pat. Nos. 4,798,793; 4,940,845 5,156,963; 5,342,768; 5,776,741 andWO89/01032.

One challenge to using a lipase for the production of fatty acid esterssuitable for biodiesel is that the price of lipase is much higher thanthe price of sodium hydroxide (NaOH) used by the strong base process.This challenge has been addressed by using an immobilized lipase, whichcan be recycled. However, the activity of the immobilized lipase must bemaintained after being recycled for a minimum number of cycles to allowa lipase-based process to compete with the strong base process in termsof the production cost. Immobilized lipases are subject to poisoning bythe lower alcohols typically used in transesterification. U.S. Pat. No.6,398,707 (issued Jun. 4, 2002 to Wu et al.) describes methods forenhancing the activity of immobilized lipases and regeneratingimmobilized lipases having reduced activity.

In particular embodiments, a recombinant lipase is expressed in the samemicroorganisms that produce the lipid on which the lipase acts. DNAencoding the lipase and selectable marker is preferably codon-optimizedcDNA. Methods of recoding genes for expression in microalgae aredescribed in U.S. Pat. No. 7,135,290.

Standards

The common international standard for biodiesel is EN 14214. ASTM D6751is the most common biodiesel standard referenced in the United Statesand Canada. Germany uses DIN EN 14214 and the UK requires compliancewith BS EN 14214.

Basic industrial tests to determine whether the products conform tothese standards typically include gas chromatography, HPLC, and others.Biodiesel meeting the quality standards is very non-toxic, with atoxicity rating (LD₅₀) of greater than 50 mL/kg.

Renewable Diesel

Renewable diesel comprises alkanes, such as C16:0 and C18:0 and thus,are distinguishable from biodiesel. High quality renewable dieselconforms to the ASTM D975 standard.

The lipids produced by the methods of the present invention can serve asfeedstock to produce renewable diesel. Thus, in another aspect of thepresent invention, a method for producing renewable diesel is provided.Renewable diesel can be produced by at least three processes:hydrothermal processing (hydrotreating); hydroprocessing; and indirectliquefaction. These processes yield non-ester distillates. During theseprocesses, triacylglycerides produced and isolated as described herein,are converted to alkanes.

In a preferred embodiment, the method for producing renewable dieselcomprises (a) cultivating a lipid-containing microorganism using methodsdisclosed herein (b) lysing the microorganism to produce a lysate, (c)isolating lipid from the lysed microorganism, and (d) deoxygenating andhydrotreating the lipid to produce an alkane, whereby renewable dieselis produced. Lipids suitable for manufacturing renewable diesel can beobtained via extraction from microbial biomass using an organic solventsuch as hexane, or via other methods, such as those described in U.S.Pat. No. 5,928,696.

In some methods, the microbial lipid is first cracked in conjunctionwith hydrotreating to reduce carbon chain length and saturate doublebonds, respectively. The material is then isomerized, also inconjunction with hydrotreating. The naptha fraction can then be removedthrough distillation, followed by additional distillation to vaporizeand distill components desired in the diesel fuel to meet a D975standard while leaving components that are heavier than desired formeeting a D 975 standard. Hydrotreating, hydrocracking, deoxygenationand isomerization methods of chemically modifying oils, includingtriglyceride oils, are well known in the art. See for example Europeanpatent applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1);EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); and U.S. Pat. Nos.7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746;5,885,440; 6,881,873.

Hydrotreating

In a preferred embodiment of the method for producing renewable diesel,treating the lipid to produce an alkane is performed by hydrotreating ofthe lipid composition. In hydrothermal processing, typically, biomass isreacted in water at an elevated temperature and pressure to form oilsand residual solids. Conversion temperatures are typically 300 to 660 F,with pressure sufficient to keep the water primarily as a liquid, 100 to170 standard atmosphere (atm). Reaction times are on the order of 15 to30 minutes. After the reaction is completed, the organics are separatedfrom the water. Thereby a distillate suitable for diesel is produced.

Hydroprocessing

A renewable diesel, referred to as “green diesel,” can be produced fromfatty acids by traditional hydroprocessing technology. Thetriglyceride-containing oils can be hydroprocessed either as co-feedwith petroleum or as a dedicated feed. The product is a diesel fuel thatconforms to the ASTM D975 specification. Thus, in another preferredembodiment of the method for producing renewable diesel, treating thelipid composition to produce an alkane is performed by hydroprocessingof the lipid composition.

In some methods of making renewable diesel, the first step of treating atriglyceride is hydroprocessing to saturate double bonds, followed bydeoxygenation at elevated temperature in the presence of hydrogen and acatalyst. In some methods, hydrogenation and deoxygenation occur in thesame reaction. In other methods deoxygenation occurs beforehydrogenation. Isomerization is then optionally performed, also in thepresence of hydrogen and a catalyst. Naphtha components are preferablyremoved through distillation. For examples, see U.S. Pat. Nos. 5,475,160(hydrogenation of triglycerides); 5,091,116 (deoxygenation,hydrogenation and gas removal); 6,391,815 (hydrogenation); and 5,888,947(isomerization).

Petroleum refiners use hydroprocessing to remove impurities by treatingfeeds with hydrogen. Hydroprocessing conversion temperatures aretypically 300 to 700 F. Pressures are typically 40 to 100 atm. Thereaction times are typically on the order of 10 to 60 minutes.

Solid catalysts are employed to increase certain reaction rates, improveselectivity for certain products, and optimize hydrogen consumption.

Hydrotreating and hydroprocessing ultimately lead to a reduction in themolecular weight of the feed. In the case of triglyceride-containingoils, the triglyceride molecule is reduced to four hydrocarbon moleculesunder hydroprocessing conditions: a propane molecule and three heavierhydrocarbon molecules, typically in the C8 to C18 range.

Indirect Liquefaction

A traditional ultra-low sulfur diesel can be produced from any form ofbiomass by a two-step process. First, the biomass is converted to asyngas, a gaseous mixture rich in hydrogen and carbon monoxide. Then,the syngas is catalytically converted to liquids. Typically, theproduction of liquids is accomplished using Fischer-Tropsch (FT)synthesis. This technology applies to coal, natural gas, and heavy oils.Thus, in yet another preferred embodiment of the method for producingrenewable diesel, treating the lipid composition to produce an alkane isperformed by indirect liquefaction of the lipid composition.

Jet Fuel

Aeroplane fuel is clear to straw colored. The most common fuel is anunleaded/paraffin oil-based fuel classified as Aeroplane A-1, which isproduced to an internationally standardized set of specifications.Aeroplane fuel is a mixture of a large number of different hydrocarbons,possibly as many as a thousand or more. The range of their sizes(molecular weights or carbon numbers) is restricted by the requirementsfor the product, for example, freezing point or smoke point.Kerosone-type Aeroplane fuel (including Jet A and Jet A-1) has a carbonnumber distribution between about 8 and 16 carbon numbers. Wide-cut ornaphta-type Aeroplane fuel (including Jet B) typically has a carbonnumber distribution between about 5 and 15 carbons.

Both Aeroplanes (Jet A and jet B) may contain a number of additives.Useful additives include, but are not limited to, antioxidants,antistatic agents, corrosion inhibitors, and fuel system icing inhibitor(FSII) agents. Antioxidants prevent gumming and usually, are based onalkylated phenols, for example, AO-30, AO-31, or AO-37. Antistaticagents dissipate static electricity and prevent sparking Stadis 450 withdinonylnaphthylsulfonic acid (DINNSA) as the active ingredient is anexample. Corrosion inhibitors, e.g., DCI-4A are used for civilian andmilitary fuels and DCI-6A is used for military fuels. FSII agents,include, e.g., Di-EGME.

A solution is blending algae fuels with existing jet fuel. The presentinvention provides such a solution. The lipids produced by the methodsof the present invention can serve as feedstock to produce jet fuel.Thus, in another aspect of the present invention, a method for producingjet fuel is provided. Herewith two methods for producing jet fuel fromthe lipids produced by the methods of the present invention areprovided: fluid catalytic cracking (FCC); and hydrodeoxygenation (HDO).

Fluid Catalytic Cracking

Fluid Catalytic Cracking (FCC) is one method which is used to produceolefins, especially propylene from heavy crude fractions. There arereports in the literature that vegetable oils such as canola oil couldbe processed using FCC to give a hydrocarbon stream useful as a gasolinefuel.

The lipids produced by the method of the present invention can beconverted to olefins. The process involves flowing the lipids producedthrough an FCC zone and collecting a product stream comprised ofolefins, which is useful as a jet fuel. The lipids produced arecontacted with a cracking catalyst at cracking conditions to provide aproduct stream comprising olefins and hydrocarbons useful as jet fuel.

In a preferred embodiment, the method for producing jet fuel comprises(a) cultivating a lipid-containing microorganism using methods disclosedherein, (b) lysing the lipid-containing microorganism to produce alysate, (c) isolating lipid from the lysate, and (d) treating the lipidcomposition, whereby jet fuel is produced.

In a preferred embodiment of the method for producing a jet fuel, thelipid composition can be flowed through a fluid catalytic cracking zone,which, in one embodiment, may comprise contacting the lipid compositionwith a cracking catalyst at cracking conditions to provide a productstream comprising C₂-C₅ olefins.

In certain embodiments of this method it may be desirable to remove anycontaminants that may be present in the lipid composition. Thus, priorto flowing the lipid composition through a fluid catalytic crackingzone, the lipid composition is pretreated. Pretreatment may involvecontacting the lipid composition with an ion-exchange resin. The ionexchange resin is an acidic ion exchange resin, such as Amberlyst™-15and can be used as a bed in a reactor through which the lipidcomposition is flowed, either upflow or downflow. Other pretreatmentsmay include mild acid washes by contacting the lipid composition with anacid, such as sulfuric, acetic, nitric, or hydrochloric acid. Contactingis done with a dilute acid solution usually at ambient temperature andatmospheric pressure.

The lipid composition, optionally pretreated, is flowed to an FCC zonewhere the hydrocarbonaceous components are cracked to olefins. Catalyticcracking is accomplished by contacting the lipid composition in areaction zone with a catalyst composed of finely divided particulatematerial. The reaction is catalytic cracking, as opposed tohydrocracking, and is carried out in the absence of added hydrogen orthe consumption of hydrogen. As the cracking reaction proceeds,substantial amounts of coke are deposited on the catalyst. The catalystis regenerated at high temperatures by burning coke from the catalyst ina regeneration zone. Coke-containing catalyst, referred to herein as“coked catalyst”, is continually transported from the reaction zone tothe regeneration zone to be regenerated and replaced by essentiallycoke-free regenerated catalyst from the regeneration zone. Fluidizationof the catalyst particles by various gaseous streams allows thetransport of catalyst between the reaction zone and regeneration zone.Methods for cracking hydrocarbons, such as those of the lipidcomposition described herein, in a fluidized stream of catalyst,transporting catalyst between reaction and regeneration zones, andcombusting coke in the regenerator are well known by those skilled inthe art of FCC processes. Exemplary FCC applications and catalystsuseful for cracking the lipid composition to produce C₂-C₅ olefins aredescribed in U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporatedin their entirety by reference.

In one embodiment, cracking the lipid composition of the presentinvention, takes place in the riser section or, alternatively, the liftsection, of the FCC zone. The lipid composition is introduced into theriser by a nozzle resulting in the rapid vaporization of the lipidcomposition. Before contacting the catalyst, the lipid composition willordinarily have a temperature of about 149 C to about 316 C (300 F to600 F). The catalyst is flowed from a blending vessel to the riser whereit contacts the lipid composition for a time of abort 2 seconds or less.

The blended catalyst and reacted lipid composition vapors are thendischarged from the top of the riser through an outlet and separatedinto a cracked product vapor stream including olefins and a collectionof catalyst particles covered with substantial quantities of coke andgenerally referred to as “coked catalyst.” In an effort to minimize thecontact time of the lipid composition and the catalyst which may promotefurther conversion of desired products to undesirable other products,any arrangement of separators such as a swirl arm arrangement can beused to remove coked catalyst from the product stream quickly. Theseparator, e.g. swirl arm separator, is located in an upper portion of achamber with a stripping zone situated in the lower portion of thechamber. Catalyst separated by the swirl arm arrangement drops down intothe stripping zone. The cracked product vapor stream comprising crackedhydrocarbons including light olefins and some catalyst exit the chambervia a conduit which is in communication with cyclones. The cyclonesremove remaining catalyst particles from the product vapor stream toreduce particle concentrations to very low levels. The product vaporstream then exits the top of the separating vessel. Catalyst separatedby the cyclones is returned to the separating vessel and then to thestripping zone. The stripping zone removes adsorbed hydrocarbons fromthe surface of the catalyst by counter-current contact with steam.

Low hydrocarbon partial pressure operates to favor the production oflight olefins. Accordingly, the riser pressure is set at about 172 to241 kPa (25 to 35 psia) with a hydrocarbon partial pressure of about 35to 172 kPa (5 to 25 psia), with a preferred hydrocarbon partial pressureof about 69 to 138 kPa (10 to 20 psia). This relatively low partialpressure for hydrocarbon is achieved by using steam as a diluent to theextent that the diluent is 10 to 55 wt-% of lipid composition andpreferably about 15 wt-% of lipid composition. Other diluents such asdry gas can be used to reach equivalent hydrocarbon partial pressures.

The temperature of the cracked stream at the riser outlet will be about510 C. to 621 C (950 F to 1150 F). However, riser outlet temperaturesabove 566 C (1050 F) make more dry gas and more olefins. Whereas, riseroutlet temperatures below 566 C (1050 F) make less ethylene andpropylene. Accordingly, it is preferred to run the FCC process at apreferred temperature of about 566 C to about 63° C., preferred pressureof about 138 kPa to about 240 kPa (20 to 35 psia). Another condition forthe process is the catalyst to lipid composition ratio which can varyfrom about 5 to about 20 and preferably from about 10 to about 15.

In one embodiment of the method for producing a jet fuel, the lipidcomposition is introduced into the lift section of an FCC reactor. Thetemperature in the lift section will be very hot and range from about700 C (1292 F) to about 760 C (1400 F) with a catalyst to lipidcomposition ratio of about 100 to about 150. It is anticipated thatintroducing the lipid composition into the lift section will produceconsiderable amounts of propylene and ethylene.

Gas and liquid hydrocarbon products produced can be analyzed by gaschromatography, HPLC, etc.

Hydrodeoxygenation

In another embodiment of the method for producing a jet fuel using thelipid composition or the lipids produced as described herein, thestructure of the lipid composition or the lipids is broken by a processreferred to as hydrodeoxygenation (HDO).

HDO means removal of oxygen by means of hydrogen, that is, oxygen isremoved while breaking the structure of the material. Olefinic doublebonds are hydrogenated and any sulphur and nitrogen compounds areremoved. Sulphur removal is called hydrodesulphurization (HDS).Pretreatment and purity of the raw materials (lipid composition or thelipids) contribute to the service life of the catalyst.

Generally in the HDO/HDS step, hydrogen is mixed with the feed stock(lipid composition or the lipids) and then the mixture is passed througha catalyst bed as a co-current flow, either as a single phase or a twophase feed stock. After the HDO/MDS step, the product fraction isseparated and passed to a separate isomerzation reactor. Anisomerization reactor for biological starting material is described inthe literature (FI 100 248) as a co-current reactor.

The process for producing a fuel by hydrogenating a hydrocarbon feed,e.g., the lipid composition or the lipids herein, can also be performedby passing the lipid composition or the lipids as a co-current flow withhydrogen gas through a first hydrogenation zone, and thereafter thehydrocarbon effluent is further hydrogenated in a second hydrogenationzone by passing hydrogen gas to the second hydrogenation zone as acounter-current flow relative to the hydrocarbon effluent. Exemplary HDOapplications and catalysts useful for cracking the lipid composition toproduce C₂-C₅ olefins are described in U.S. Pat. No. 7,232,935, which isincorporated in its entirety by reference.

Typically, in the hydrodeoxygenation step, the structure of thebiological component, such as the lipid composition or lipids herein, isdecomposed, oxygen, nitrogen, phosphorus and sulphur compounds, andlight hydrocarbons as gas are removed, and the olefinic bonds arehydrogenated. In the second step of the process, i.e. in the so-calledisomerization step, isomerzation is carried out for branching thehydrocarbon chain and improving the performance of the paraffin at lowtemperatures.

In the first step i.e. HDO step of the cracking process, hydrogen gasand the lipid composition or lipids herein which are to be hydrogenatedare passed to a HDO catalyst bed system either as co-current orcounter-current flows, said catalyst bed system comprising one or morecatalyst bed(s), preferably 1-3 catalyst beds. The HDO step is typicallyoperated in a co-current manner. In case of a HDO catalyst bed systemcomprising two or more catalyst beds, one or more of the beds may beoperated using the counter-current flow principle.

In the HDO step, the pressure varies between 20 and 150 bar, preferablybetween 50 and 100 bar, and the temperature varies between 200 and 500C, preferably in the range of 300-400 C.

In the HDO step, known hydrogenation catalysts containing metals fromGroup VII and/or VIB of the Periodic System may be used. Preferably, thehydrogenation catalysts are supported Pd, Pt, Ni, NiMo or a CoMocatalysts, the support being alumina and/or silica. Typically,NiMo/Al₂O₃ and CoMo/Al₂O₃ catalysts are used.

Prior to the HDO step, the lipid composition or lipids herein mayoptionally be treated by prehydrogenation under milder conditions thusavoiding side reactions of the double bonds. Such prehydrogenation iscarried out in the presence of a prehydrogenation catalyst attemperatures of 50 400 C and at hydrogen pressures of 1 200 bar,preferably at a temperature between 150 and 250 C and at a hydrogenpressure between 10 and 100 bar. The catalyst may contain metals fromGroup VIII and/or VIB of the Periodic System. Preferably, theprehydrogenation catalyst is a supported Pd, Pt, Ni, NiMo or a CoMocatalyst, the support being alumina and/or silica.

A gaseous stream from the HDO step containing hydrogen is cooled andthen carbon monoxide, carbon dioxide, nitrogen, phosphorus and sulphurcompounds, gaseous light hydrocarbons and other impurities are removedtherefrom. After compressing, the purified hydrogen or recycled hydrogenis returned back to the first catalyst bed and/or between the catalystbeds to make up for the withdrawn gas stream. Water is removed from thecondensed liquid. The liquid is passed to the first catalyst bed orbetween the catalyst beds.

After the HDO step, the product is subjected to an isomerization step.It is substantial for the process that the impurities are removed ascompletely as possible before the hydrocarbons are contacted with theisomerization catalyst. The isomerization step comprises an optionalstripping step, wherein the reaction product from the HDO step may bepurified by stripping with water vapor or a suitable gas such as lighthydrocarbon, nitrogen or hydrogen. The optional stripping step iscarried out in counter-current manner in a unit upstream of theisomerization catalyst, wherein the gas and liquid are contacted witheach other, or before the actual isomerization reactor in a separatestripping unit utilizing counter-current principle.

After the stripping step the hydrogen gas and the hydrogenated lipidcomposition or lipids herein, and optionally an n-paraffin mixture, arepassed to a reactive isomerization unit comprising one or severalcatalyst bed(s). The catalyst beds of the isomerization step may operateeither in co-current or counter-current manner.

It is important for the process that the counter-current flow principleis applied in the isomerization step. In the isomerization step this isdone by carrying out either the optional stripping step or theisomerization reaction step or both in counter-current manner.

The isomerization step and the HDO step may be carried out in the samepressure vessel or in separate pressure vessels. Optionalprehydrogenation may be carried out in a separate pressure vessel or inthe same pressure vessel as the HDO and isomerization steps.

In the isomerization step, the pressure varies in the range of 20 150bar, preferably in the range of 20 100 bar, the temperature beingbetween 200 and 500 C, preferably between 300 and 400 C.

In the isomerization step, isomerization catalysts known in the art maybe used. Suitable isomerization catalysts contain molecular sieve and/ora metal from Group VII and/or a carrier. Preferably, the isomerizationcatalyst contains SAPO-11 or SAPO41 or ZSM-22 or ZSM-23 or ferrieriteand Pt, Pd or N1 and Al₂O₃ or SiO₂. Typical isomerization catalysts are,for example, Pt/SAPO-11/Al₂O₃, Pt/ZSM-22/Al₂O₃, Pt/ZSM-23/Al₂O₃ andPt/SAPO-11/SiO₂.

As the product, a high quality hydrocarbon component of biologicalorigin, useful as a diesel fuel or a component thereof, is obtained, thedensity, cetane number and performance at low temperate of saidhydrocarbon component being excellent.

Microbe Engineering

As noted above, in certain embodiments of the present invention it isdesirable to genetically modify a microorganism to enhance lipidproduction, modify the properties or proportions of components generatedby the microorganism, or to improve or provide de novo growthcharacteristics on a variety of feedstock materials.

Promoters, cDNAs, and 3′UTRs, as well as other elements of the vectors,can be generated through cloning techniques using fragments isolatedfrom native sources (see for example Molecular Cloning: A LaboratoryManual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press; andU.S. Pat. No. 4,683,202). Alternatively, elements can be generatedsynthetically using known methods (see for example Gene. 1995 Oct. 16;164(1):49-53). Microbial engineering methods are generally known in theart, e.g., U.S. Pat. App. No. 20090011480, herein incorporated byreference in its entirety, for all purposes.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1 Biomass Increase by Low Intensity-Illumination Application toMicroalgae Fermentation

Botryococcus naturally synthesizes and tolerates hydrocarbon mixturesand produces as much as 85% hydrocarbon by weight, and in many cases themajor hydrocarbon is botryococcenes. It is also known to be an obligatephototroph, but it seems to have the ability to uptake glucose(Reference: “Biosynthesis of the triterpenoids, botryococcenes andtetramethylsqualene in the B race of Botryococcus braunii via thenon-mevalonate pathway” Sato et al. 2003. Tetrahedron Letter44:7035-7037).

Botryococcus culture is grown on BG11 media (Reference: “Autotrophiccultivation of Botryococcus braunii for the production of hydrocarbonsand exopolysaccharides in various media”. Dayananda et al. 2007. Biomassand Bioenergy. 31: 87-93) at 25-35° C. in a bioreactor with 10-30% ofdissolved oxygen. The effects of light signal on heterotrophic growth ofBotryococcus are tested by comparing the cellular dry weight of thecultures from different conditions (dark+no glucose, dark+glucose,light+no glucose, and light+glucose). Optimum light intensity (0.01-300μmol photons m⁻²s⁻¹) and different light spectrum (360-700 nm) as wellas different light periods (9-16 hr) are tested. Combination of lowirradiance of light and glucose results in a) improved growth rate, b)increased products such as carotenoids, lipids, and botryococcenes.

Example 2 Light Regulation of Isoprenoid Pathway

Isoprene, monoterpenes and sesquiterpenes are synthesized and emitted bysome plant and microalgal species, but not all species have thisability. These volatile, nonessential isoprenoid compounds share thesame biochemical precursors as larger commercially useful isoprenoidssuch as carotenoids and hydrocarbons. Two separate pathways operate inplant cells to synthesize prenyl diphosphate precursors common to allisoprenoids.

Cytosolic and mitochondrial precursors are produced by the mevalonicacid (MVA) pathway whereas the recently discovered methylerythritolphosphate (MEP) pathway is located in plastids. Botryococcus brauniiproduces hydrocarbon by the non-mevalonic, MEP pathway (FIG. 2).

Light is the most important environmental factor for regulation of MEPpathway. 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is therate limiting step, and the expression of the gene encoding DXR isregulated by light (Reference: “Expression and molecular analysis of theArabidopsis DXR gene encoding 1-deoxy-d-xylulose 5-phosphatereductoisomerase, the first committed enzyme of the2-c-methyl-d-erythritol 4-phosphate pathway”. Carretero-Paulet et al.Plant Physiology. 2002. 129:1581-1591).

Another example is blue-light activation of genes encoding carotenoidbiosynthetic enzymes in Chlamydomonas reinhardtii, an unicellular greenalga. Microarray and quantitative PCR experiments showed the genesencoding carotenoid biosynthetic enzymes such as PDS, HDS, PSY, and ZDSare activated by very low irradiance of white light (0.01 μmol photonsm⁻²s⁻¹) and blue light. Further evidence suggested phototropin, a bluelight receptor, is involved in blue-light activated gene expression forcarotenoid biosynthesis (Reference: “Phototropin involvement in theexpression of genes encoding chlorophyll and carotenoid biosynthesisenzymes and LHC apoproteins in Chlamydomonas reinhardtii”. Im et al. ThePlant Journal. 2006. 48:1-16).

One example is the increase of hydrocarbon production from Botryococcus.Various light intensity (0.01-300 μmol photons m⁻²s⁻¹) and differentlight spectrum (360-700 nm) are applied to a Botryococcus culture, andthe amount of different hydrocarbon species are measured by GC-MS.

Example 3 Growth of Neochloris oleabundans Under Different LightConditions

Materials and Methods

Microalgae and Culture Condition

Neochloris oleabundans strain UTEX 1185 was obtained from the culturecollection of algae at the University of Texas (Austin, Tex. USA).Initial culture of the microalgae was grown in Erlenmeyer 250 ml flaskcontaining 120 ml modified bold 3 N medium with 2% glucose at 25° C.room temperature with an aluminum foil loosely covering the flask on anorbital shaker at 130 rpm under alternating two 40 W natural sunshine(392316, Philips) and two 40 W plant and aquarium (392282, Philips)fluorescent light bulbs. The culture medium (modified MB3N) containedthe following components per 1 L of deionized water: 0.75 g NaNO₃, 0.075g K₂HPO₄, 0.074 g MgSO₄ 7H₂O, 0.025 g CaCl₂ 2H₂O, 0.176 g KH₂PO₄, 0.025g NaCl, 6 ml of P-IV metal solution (0.75 g Na₂EDTA 2H₂O, 0.097 g FeCl₃6H₂O, 0.041 g MnCl₂ 4H₂O, 0.005 g ZnCl₂, 0.002 g CoCl₂ 6H₂O, 0.004 gNa₂MoO4 2H₂O in 1 L dI water), 1 ml of each three vitamins (0.1 mMvitamin B12, 0.1 mM biotin, 6.5 mM thiamine dissolved separately in 50mM HEPES pH7.8). Final pH of the medium was adjusted to 7.5 with 20% KOHbefore autoclaving the medium. The vitamin solutions were added to cooldown the autoclaved medium. Once the initial culture reached certainconfluence, its concentration was measured using optical density (OD) at680 nm and 750 nm using Genesys 10 UV spectrophotometer (ThermoScientific).

Experimental Procedure and Growth Measurement

Three different wavelengths of light (white, blue, and red) were tested.LED lights were purchased from Super Bright LEDs, Inc. (white:RL5-W3030, blue: RL5-B2430, red: RL5—R1330). For each light wavelength,four different conditions were set up in duplicate as follows:

1-2. Modified MB3N+no glucose+dark

3-4. Modified MB3N+no glucose+dim light

5-6. Modified MB3N+2% glucose+dark

7-8. Modified MB3N+2% glucose+dim light

A total of eight 250 ml Erlenmeyer flasks containing a final volume of120 ml cell culture were prepared with an initial cell concentration ofOD 0.1 at 750 nm (˜1.1×10⁶ cells/ml) for each condition. Intensity oflight was set at 3-4 μmol/m²s⁻¹ photons for white, 2-3 μmol/m²s⁻¹photons for blue, and 1-2 μmol/m²s⁻¹ photons for red. The speed of theorbital shaker was set at 135 rpm. The experiment was carried out atroom temperature for two weeks. 1 ml of cell culture was obtained fromeach flask every 24 hrs to evaluate cell concentrations by measuring ODat 680 nm and 750 nm using Genesys 10 UV spectrophotometer from ThermoFisher Scientific (Waltham, Mass. USA). Specific growth rate wasdetermined by plotting the logarithm of culture optical density againsttime (FIG. 3). The combination of a low irradiance of red, white, orblue light and glucose resulted in an improved growth rate compared tocontrols.

Example 4 Growth of Botryococcus sudeticus Under Different LightConditions

Materials and Methods

Strains and Media

Botryococcus sudeticus strain UTEX 2629 was obtained from the algaeculture collection at the University of Texas (Austin, Tex. USA). Stockculture was grown in Erlenmeyer 250 ml flasks containing 120 ml modifiedBG11 medium with 2% glucose at 25° C. room temperature with dim light(4-5 μmol/m²s⁻¹ photons) on an orbital shaker at 130 rpm. Dim lightingis composed of two different bulbs, 40 W natural sunshine (392316Philips) and 40 W plant and aquarium fluorescent light bulbs (392282Philips). 1 L of culture medium (Modified BG-11) contained: 10 mM HEPES(pH 7.8), 1.5 g NaNO₃, 0.04 g K₂HPO₄, 0.06 g MgSO₄ 7H₂O, 0.036 g CaCl₂2H₂O, 0.006 g Citric acid H₂O, 0.0138 g Ammonium Ferric Citrate, 0.001 gNa₂EDTA 2H₂O, 0.02 g Na₂CO₃, 2.86 mg H₃BO₃, 1.81 mg MnCl₂ 4H₂O, 0.22 mgZnSO₄ 7H₂O, 0.39 mg Na₂MoO₄ 2H₂O, 0.079 mg CuSO₄5H₂O, 0.0494 mg Co(NO₃)₂6H₂O, 0.5 g casein hydrolysate, and 1 ml of each three vitamins (0.1 mMvitamin B12, 0.1 mM biotin, 6.5 mM thiamine dissolved separately in 50mM HEPES pH7.8). Final pH of the medium was adjusted to 7.8 with 20%KOH.

Experimental Procedure and Growth Measurement

Three different wavelengths of light (white, blue, and red) were tested.LED lights were purchased from Super Bright LEDs, Inc. (white:RL5-W3030, blue: RL5-B2430, red: RL5—R1330). For each light wavelength,four different conditions were set up in duplicate as follows:

1-2. Modified BG-11+no glucose+dark

3-4. Modified BG-11+no glucose+dim light

5-6. Modified BG-11+2% glucose+dark

7-8. Modified BG-11+2% glucose+dim light

A total of eight 250 ml Erlenmeyer flasks containing a final volume of120 ml cell culture were prepared with an initial cell concentration ofOD 0.1 at 750 nm (˜1.1×10⁶ cells/ml) for each condition. Intensity oflight was set at 3-4 μmol/m²s⁻¹ photons for white, 2-3 μmol/m²s⁻¹photons for blue, and 1-2 μmol/m²s⁻¹ photons for red. The speed of theorbital shaker was set at 135 rpm. The experiment was carried out atroom temperature for two weeks. 1 ml of cell cultures was obtained fromeach flask everyday to evaluate cell concentrations by measuring OD at680 nm and 750 nm using Genesys 10 UV spectrophotometer from ThermoFisher Scientific (Waltham, Mass. USA). Specific growth rate wasdetermined by plotting the logarithm of culture optical density againsttime (FIG. 4). The combination of a low irradiance of red, white, orblue light and glucose resulted in an improved growth rate compared tocontrols.

Example 5 Botryococcus braunii: Fermentation with ControlledIllumination

Materials and Methods

Strains and Media

Botryococcus braunii strain UTEX 2441 was obtained from the algaeculture collection at the University of Texas (Austin, Tex. USA). Stockculture was grown in Erlenmeyer 250 ml flasks containing 120 ml modifiedBG11 medium with 2% glucose at 25° C. room temperature with dim light(4-5 μmol/m²s⁻¹ photons) on an orbital shaker at 130 rpm. Dim lightingwas composed of two different bulbs, 40 W natural sunshine (392316Philips) and 40 W plant and aquarium fluorescent light bulbs (392282Philips). 1 L of culture medium (Modified BG-11) contained: 10 mM HEPES(pH 7.8), 1.5 g NaNO₃, 0.04 g K₂HPO₄, 0.06 g MgSo₄ 7H₂O, 0.036 g CaCl₂2H₂O, 0.006 g Citric acid H₂O, 0.0138 g Ammonium Ferric Citrate, 0.001 gNa₂EDTA 2H₂O, 0.02 g Na₂CO₃, 2.86 mg H₃BO₃, 1.81 mg MnCl₂ 4H₂O, 0.22 mgZnSO₄ 7H₂O, 0.39 mg Na₂MoO₄ 2H₂O, 0.079 mg Cu50₄5H₂O, 0.0494 mg Co(NO₃)₂6H₂O, 0.5 g casein hydrolysate, and 1 ml of each three vitamins (0.1 mMvitamin B12, 0.1 mM biotin, 6.5 mM thiamine dissolved separately in 50mM HEPES pH7.8). Final pH of the medium was adjusted to 7.8 with 20%KOH.

Experimental Procedure and Growth Measurement

Three different wavelengths of lights (white, blue, and red) weretested. LED lights were purchased from Super Bright LEDs, Inc. (white:RL5-W3030, blue: RL5-B2430, red: RL5—R1330). For each light wavelength,four different conditions were set up in duplicate as follows:

1-2 Modified BG-11+no glucose+dark

3-4 Modified BG-11+no glucose+dim light

5-6 Modified BG-11+2% glucose+dark

7-8 Modified BG-11+2% glucose+dim light

A total of eight 250 ml Erlenmeyer flasks containing a final volume of120 ml cell culture were prepared with an initial cell concentration ofO.D 0.1 at 750 nm (˜1.1×10⁶ cells/ml) for each condition. The intensityof light was set at 3-4 μmol/m²s⁻¹ photons for white, 2-3 μmol/m²s⁻¹photons for blue, and 1-2 μmol/m²s⁻¹ photons for red. The speed of theorbital shaker was set at 150 rpm. The experiment was carried out atroom temperature for two weeks. 5 ml of each cell culture was obtainedfrom each flask every two days to evaluate cell growth by dry cellweight (DCW). Specific growth rate was determined by plotting theculture DCW against time (FIG. 5).

Fluorescence Measurement of Neutral Lipid by Using Nile Red

In 1 ml of algal suspension, 4 ul of Nile Red solution in acetone (250ug/ml) was added. The mixture was vortexed 2 times during a 10 minuteincubation at room temperature. After incubation, 200 ul of stainedalgal samples were transferred into individual wells in a 96-well plate.Fluorescence was measured on a Molecular Devices 96 well platespectrofluorometer with a 490 nm excitation and 585 nm emissionwavelength with 530 emission filter cut off. In order to determine therelative fluorescence intensity of algal samples, blank (Nile Red alonein the medium) was subtracted from the fluorescence intensity.

Results

Red light+glucose increased the growth rate of UTEX 2441 by 35% comparedto heterotrophic culture in the dark (Dark+glu) (FIG. 5). Lipid levelsalso increased by 52% under red light conditions compared to controls(FIG. 6).

Example 6 Chlamydomonas reinhardtii: Fermentation with ControlledIllumination

Materials and Methods

Strains and Media

Chlamydomonas reinhardtii strain UTEX 2243 was obtained from the algaeculture collection at the University of Texas (Austin, Tex. USA). Stockcultures were grown separately in Erlenmeyer 250 ml flasks containing120 ml TAP medium at 25° C. room temperature with dim light (4-5μmol/m²s⁻¹ photons) on an orbital shaker at 130 rpm. Dim lighting wascomposed of two different bulbs (40 W natural sunshine (392316 Philips)and 40 W plant and aquarium fluorescent light bulbs (392282 Philips)). 1L of culture medium (TAP) contained: 2.42 g Tris, 25 ml of TAP saltssolution (15 g NH4Cl, 4 g MgSO₄ 7H₂O, 2 g CaCl₂ 2H₂O), 0.375 mlPhosphate solution (28.8 g K₂HPO₄, 14.4 g KH2PO₄ in 100 ml of water), 1ml Hutner's trace elements solution (1 L of Trace metal solutioncontains 50 g EDTA disodium Salt, 22 g ZnSO₄ 7H₂O, 11.4 g H₃BO₄, 5.06 gMnCl₂ 4H₂O, 1.61 g CoCl₂ 6H₂O, 1.57 g CuSO₄ 5H₂O, 1.10 (NH₄)₆Mo₇O₂₄4H₂O, 4.99 g FeSO₄ 7H₂O with pH 7.0 using either KOH or HCl) and 1 ml ofglacial acetic acid. Final pH of medium is 7.0 adjusted with glacieracetic acid. Tris minimal medium (TP) was made with all componentslisted above except acetic acid. The medium's pH was adjusted to 7.0with HCl.

Experimental Procedure and Growth Measurement

LED lights were purchased from Super Bright LEDs, Inc. (RL5-W3030). Fourdifferent conditions were set up in duplicate as follows:

1-2. TP (no acetic acid)+dark

3-4. TP (no acetic acid)+dim light

5-6. TAP (acetic acid)+dark

7-8. TAP (acetic acid)+dim light

A total of eight 250 ml Erlenmeyer flasks containing a final volume of120 ml cell culture were prepared with an initial cell concentration of1.0×10⁵ cells/ml for each condition. The intensity of light was set at3-5 μmol/m²s⁻¹ photons. The speed of the orbital shaker was set at 140rpm. The experiment was carried out at room temperature for one week.500 ul of cell culture was obtained from each flask every day toevaluate cell growth by counting cell numbers. Cells were deflagellatedusing lugol solution (1:20) prior to counting. Specific growth rate wasdetermined by plotting the cell number against time. The combination ofa low irradiance of white light and TAP resulted in an improved growthrate compared to controls (FIG. 7).

Example 7 Cultivation of Microalgae with a Low Irradiance of Light

Materials and Methods

Strains and Media

Microalgae strains (e.g., Chlamydomonas, Botryococcus, Neochloris,Cyanophyta, Chlorophyta, Rhodophyta, Cryptophyta, Chlorarachniophyta,Haptophyta, Euglenophyta, Heterokontophyta, Diatoms and/or thosedescribed in the description above) are obtained from, e.g., the algaeculture collection at the University of Texas (Austin, Tex. USA). Stockculture is grown, e.g., in Erlenmeyer 250 ml flasks containing theappropriate medium (see, e.g., manufacturer's instructions) at about 25°C. room temperature with dim light (e.g., 4-5 μmol/m²s⁻¹ photons) on anorbital shaker at about 130 rpm. An appropriate carbon source is used inthe culture media, e.g., glucose. Dim lighting can be composed of twodifferent bulbs, e.g., 40 W natural sunshine (392316 Philips) and 40 Wplant and aquarium fluorescent light bulbs (392282 Philips). The finalpH of the medium is adjusted as appropriate for the particular strain.See, e.g., manufacturer's instructions.

Experimental Procedure and Growth Measurement

Three different wavelengths of light (white, blue, and red) are tested.LED lights are purchased from, e.g., Super Bright LEDs, Inc. (white:RL5-W3030, blue: RL5-B2430, red: RL5—R1330). For each light wavelength,four different conditions are set up in duplicate as follows:

1-2 no carbon+dark

3-4 no carbon+dim light

5-6 carbon+dark

7-8 carbon+dim light

A total of eight 250 ml Erlenmeyer flasks containing a final volume of120 ml cell culture are prepared with an initial cell concentration of,e.g., O.D 0.1 at 750 nm (˜1.1×10⁶ cells/ml) for each condition. Optimumlight intensity (e.g., 0.01-300 μmol photons m⁻²s⁻¹) and different lightspectrums (e.g., 360-700 nm) as well as different light periods (e.g.,9-16 hr of light) are tested. The intensity of light is set at, e.g.,3-4 μmol/m²s⁻¹ photons for white, 2-3 μmol/m²s⁻¹ photons for blue, and1-2 μmol/m²s⁻¹ photons for red. Various carbon sources at variousconcentrations are tested, e.g., glucose, sucrose, fructose at aconcentration of, e.g., 1%, 2%, or 3% of culture media. The speed of theorbital shaker is set at, e.g., 150 rpm. The experiment is carried outat room temperature for less than one, one, two, three, or more weeks.An aliquot of each cell culture is obtained from each flask every one totwo days to evaluate cell growth by, e.g., dry cell weight (DCW).Specific growth rate is determined by plotting the culture DCW againsttime.

Measurement of Material of Interest

The amount of material of interest (hydrocarbon, lipid, etc.) in themedia is measured using standard means known in the art, e.g., GC-MS orNile Red as described above. For example, in 1 ml of algal suspension, 4ul of Nile Red solution in acetone (250 ug/ml) is added. The mixture isvortexed during incubation at room temperature. After incubation,100-200 ul of stained algal samples are transferred into individualwells in a 96-well plate. Fluorescence is measured on, e.g., a MolecularDevices 96 well plate spectrofluorometer with a 490 nm excitation and585 nm emission wavelength with 530 emission filter cut off. In order todetermine the relative fluorescence intensity of algal samples, blank(Nile Red alone in the medium) is subtracted from the fluorescenceintensity.

Results

Red, white, and/or blue light in combination with a carbon sourceincrease the growth rate of the microalgae strain compared to controls.Material of interest (e.g., hydrocarbon or lipid) levels produced by theexperimental microalgae strain (red, white, and/or blue light incombination with a carbon source) increase compared to controls.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

1. A method for cultivating a microalgae capable of heterotrophicgrowth, comprising: incubating the microalgae under a heterotrophicgrowth condition for a period of time sufficient to allow the microalgaeto grow, wherein the heterotrophic growth condition comprises a mediacomprising a carbon source, and wherein the heterotrophic growthcondition further comprises a low irradiance of light.
 2. The method ofclaim 1, wherein the microalgae is a Botryococcus strain, wherein thecarbon source is glucose, and wherein the low irradiance of light isbetween 1-10 μmol photons m⁻²s⁻¹.
 3. The method of claim 1, wherein themicroalgae is a Botryococcus sudeticus strain, a Botryococcus strain, aUTEX 2629 strain, a Botryococcus braunii strain, a UTEX 2441 strain, aNeochloris oleabundans strain, a Neochloris strain, a UTEX 1185 strain,a Chlamydomonas reinhardtii strain, a Chlamydomonas strain, a UTEX 2243strain, or a strain comprising a photoreceptor.
 4. (canceled)
 5. Themethod of claim 2, wherein the microalgae is a UTEX 2629 strain or aUTEX 2441 strain.
 6. (canceled)
 7. (canceled)
 8. (canceled) 9.(canceled)
 10. (canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled)14. (canceled)
 15. The method of claim 1, wherein the carbon source isglucose.
 16. The method of claim 1, wherein the carbon source isselected from the group consisting of a fixed carbon source, glucose,fructose, sucrose, galactose, xylose, mannose, rhamnose,N-acetylglucosamine, glycerol, floridoside, glucuronic acid, cornstarch, depolymerized cellulosic material, sugar cane, sugar beet,lactose, milk whey, and molasses.
 17. The method of claim 1, wherein thelight is produced by a natural light source, the light is natural sunlight, the light comprises full spectrum light or a specific wavelengthof light, the light is produced by an artificial light source, or thelight is artificial light.
 18. (canceled)
 19. (canceled)
 20. (canceled)21. (canceled)
 22. The method of claim 1, wherein the intensity of thelow irradiance of light is between 0.01-1 μmol photons m⁻²s⁻¹, between1-10 μmol photons m⁻²s⁻¹, between 10-100 μmol photons m⁻²s⁻¹, between100-300 μmol photons m⁻²s⁻¹, 3-4 μmol/m²s⁻¹ photons, 2-3 μmol/m²s⁻¹photons, 1-2 μmol/m²s⁻¹ photons, or 3-5 μmol/m²s⁻¹ photons. 23.(canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)28. The method of claim 1, further comprising producing a material fromthe microalgae.
 29. The method of claim 28, wherein the material is apolysaccharide, a pigment, a lipid, or a hydrocarbon.
 30. The method ofclaim 28, wherein the material is a hydrocarbon.
 31. The method of claim28, further comprising recovering the material.
 32. The method of claim28, further comprising extracting the material.
 33. The method of claim28, further comprising processing the material.
 34. The method of claim31, further comprising processing the material.
 35. The method of claim33, wherein the processing of the material produces a processedmaterial.
 36. The method of claim 35, wherein the processed material isselected from the group consisting of a fuel, biodiesel, jet fuel, acosmetic, a pharmaceutical agent, a surfactant, and a renewable diesel.37. The method of claim 1, wherein the growth rate of the microalgae ishigher than a second microalgae incubated under a second heterotrophicgrowth condition for a period of time sufficient to allow the microalgaeto grow, wherein the second heterotrophic growth condition comprises agrowth media comprising a carbon source, and wherein the secondheterotrophic growth condition does not comprise a low irradiance oflight.
 38. (canceled)
 39. A method of manufacturing a material,comprising: providing a microalgae capable of producing the material;culturing the microalgae in a media, wherein the media comprises acarbon source; applying a low irradiance of light to the microalgae; andallowing the microalgae to accumulate at least 10% of its dry cellweight as the material.
 40. (canceled)
 41. A bioreactor system,comprising: a bioreactor; a culture media comprising a carbon source,wherein the culture media is located inside the bioreactor; a microalgaeadapted for heterotrophic growth, wherein the microalgae is located inthe culture media; and a light source, wherein the light source producesa low irradiance of light, and wherein the light source is operativelycoupled to the bioreactor.
 42. (canceled)
 43. (canceled)
 44. (canceled)45. (canceled)
 46. (canceled)
 47. (canceled)